Inhibitors of thrombin

ABSTRACT

This invention relates to novel biologically active molecules which bind to and inhibit thrombin. Specifically, these molecules are characterized by a thrombin anion-binding exosite association moiety (ABEAM); a linker portion of at least 18Å in length; and a thrombin catalytic site-directed moiety (CSDM). This invention also relates to compositions, combinations and methods which employ these molecules for therapeutic, prophylactic and diagnostic purposes.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.07/549,388, filed Jul. 6, 1990, now U.S. Pat. No. 5,196,404, issued Mar.23, 1993, which is a continuation-in-part of U.S. application Ser. No.07/395,482, filed Aug. 18, 1989, now abandoned.

TECHNICAL FIELD OF INVENTION

This invention relates to novel biologically active molecules which bindto and inhibit thrombin. Specifically, these molecules are characterizedby a thrombin anion-binding exosite associating moiety (ABEAM); a linkerportion of at least 18Å in length; and a thrombin catalyticsite-directed moiety (CSDM). This invention also relates tocompositions, combinations and methods which employ these molecules fortherapeutic, prophylactic and diagnostic purposes.

BACKGROUND ART

Acute vascular diseases, such as myocardial infarction, stroke,pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion,and other blood system thromboses constitute major health risks. Suchdiseases are caused by either partial or total occlusion of a bloodvessel by a blood clot, which contains fibrin and platelets.

Current methods for the treatment and prophylaxis of thrombotic diseasesinvolve therapeutics which act in one of two different ways. The firsttype of therapeutic inhibits thrombin activity or thrombin formation,thus preventing clot formation. These drugs also inhibit plateletactivation and aggregation. The second category of therapeuticaccelerates thrombolysis and dissolves the blood clot, thereby removingit from the blood vessel and unblocking the flow of blood [J. P.Cazenave et al., Agents Action, 15, Suppl., pp. 24-49 (1984)].

Heparin, a compound of the former class, has been widely used to treatconditions, such as venous thromboembolism, in which thrombin activityis responsible for the development or expansion of a thrombus. Althougheffective, heparin produces many undesirable side effects, includinghemorrhaging and thrombocytopenia. This has led to a search for a morespecific and less toxic anticoagulant.

Hirudin is a naturally occurring polypeptide which is produced by theblood sucking leech Hirudo medicinalis. This compound, which issynthesized in the salivary gland of the leech, is the most potentnatural inhibitor of coagulation known. Hirudin prevents blood fromcoagulating by binding tightly to thrombin (K_(d) =2⁻¹¹ M) in a 1:1stoichiometric complex [S. R. Stone and J. Hofsteenge, "Kinetics of theInhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28(1986)]. This, in turn, inhibits thrombin from catalyzing the conversionof fibrinogen to fibrin (clot), as well as inhibiting all otherthrombin-mediated processes [J. W. Fenton, II, "Regulation of ThrombinGeneration and Functions", Semin. Thromb, Hemost., 14, pp. 234-40(1988)].

The actual binding between hirudin and thrombin is a two-step process.Initially, hirudin binds to a "low" affinity site on the thrombinmolecule (K_(d) =1×10⁻⁸ M) which is separate from the catalytic site.This binding involves interaction of structure from the C-terminus ofhirudin with an "anion-binding exosite" (ABE) in thrombin [J. W. Fenton,II et al., "Thrombin Anion Binding Exosite Interactions with Heparin andVarious Polyanions", Ann New York Acad. Sci., 556, pp. 158-65 (1989)].Following the low affinity binding, the hirudinthrombin complexundergoes a conformational change and hirudin then binds to the "high"affinity site on thrombin IS. Kono et al., "Analysis of SecondaryStructure of Hirudin and the Conformational Change Upon Interaction withThrombin" Arch Biochem Biophys., 267, pp. 158-66 (1988)]. This lattersite corresponds to the active site of thrombin.

The isolation, purification and chemical composition of hirudin areknown in the art [P. Walsmann and F. Markwardt, "Biochemical andPharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie,36, pp. 653-60 (1981)]. More recently, the complete amino acid sequenceof the polypeptide has been elucidated [J. Dodt et al., "The CompleteCovalent Structure of Hirudin: Localization of the Disulfide Bonds"Biol. Chem. Hoppe-Seyler, 366, pp. 379-85 (1985); S. J. T. Mao et al.,"Rapid Purification and Revised Amino Terminal Sequence of Hirudin: ASpecific Thrombin Inhibitor of the Blood-Sucking Leech" Anal Biochem,161, pp 514-18 (1987); and R. P. Harvey et al., "Cloning and Expressionof a cDNA Coding for the Anti-Coagulant Hirudin from the BloodsuckingLeech, Hirudo medicinalis", Proc. Natl. Acad. Sci. USA, 83, pp. 1084-88(1986)].

At least ten different isomorphic forms of hirudin have been sequencedand have been shown to differ slightly in amino acid sequence [D.Tripier, "Hirudin: A Family of Iso-Proteins. Isolation and SequenceDetermination of New Hirudins", Folia Haematol., 115, pp. 30-35 (1988)].All forms of hirudin comprise a single polypeptide chain proteincontaining 65 or 66 amino acids in which the amino terminus primarilycomprises hydrophobic amino acids and the carboxy terminus typicallycomprises polar amino acids. More specifically, all forms of hirudin arecharacterized by an N-terminal domain (residues 1-39) stabilized bythree disulfide bridges in a 1-2, 3-5, and 4-6 half-cysteinyl patternand a highly acidic C-terminal segment (residues 40-65). In addition,the C-terminal segment of hirudin is characterized by the presence of atyrosine residue at amino acid position 63 which is sulfated.

In animal studies, hirudin, purified from leeches, has demonstratedefficacy in preventing venous thrombosis, vascular shunt occlusion andthrombin-induced disseminated intravascular coagulation. In addition,hirudin exhibits low toxicity, little antigenicity and a very shortclearance time from circulation [F Markwardt et al., "PharmacologicalStudies on the Antithrombotic Action of Hirudin in Experimental Animals"Thromb Haemost, 47, pp 226-29 (1982)].

In an effort to create a greater supply of hirudin, attempts have beenmade to produce the polypeptide through recombinant DNA techniques, Thepresence of an O-sulfated tyrosine residue on native hirudin and theinability of microorganisms to perform a similar protein modificationmade the prospect of recombinant production of biologically activehirudin highly speculative. The observation that desulfatohirudins werealmost as active as their sulfated counterparts [U.S. Pat. No.4,654,302], however, led the way to the cloning and expression ofhirudin in E. coli [European patent applications 158,564, 168,342 and171,024] and yeast [European patent application 200,655]. Despite theseadvances, hirudin is still moderately expensive to produce and it is notwidely available commercially.

Recently, efforts have been made to identify peptide fragments of nativehirudin which are also effective in prolonging clotting times. Anunsulfated 21 amino acid C-terminal fragment of hirudin,N-acetylhirudin₄₅₋₆₅, inhibits clot formation in vitro. In addition,several other smaller, unsulfated peptides corresponding to theC-terminal 11 or 12 amino acids of hirudin (residues 55-65 and 54-65)have also demonstrated efficacy in inhibiting clot formation in vitro[J. L. Krstenansky et al., "Antithrombin Properties of C-terminus ofHirudin Using Synthetic Unsulfated N-acetyl-hirudin₄₅₋₆₅ ", FEBS Lett,211, pp. 10-16 (1987)]. Such peptide fragments, however, may not befully satisfactory to dissolve blood clots in on-going therapy regimensbecause of low activity. For example, N-acetyl-hirudin₄₅₋₆₅ has aspecific activity four orders of magnitude lower than native hirudin.

In addition to catalyzing the formation of a fibrin clot, thrombin hasseveral other bioregulatory roles [J. W. Fenton, II, "ThrombinBioregulatory Functions", Adv. Clin. Enzymol, 6, pp. 186-93 (1988)]. Forexample, thrombin directly activates platelet aggregation and releasereactions. This means that thrombin plays a central role in acuteplatelet-dependent thrombosis [S. R. Hanson and L. A. Harker,"Interruption of Acute Platelet-Dependent Thrombosis by the SyntheticAntithrombin D-Phenylalanyl-L-Prolyl-L-Arginylchloromethylketone", Proc.Natl Acad. Sci. USA, 85, pp. 3184-88 (1988)]. Thrombin can also directlyactivate an inflammatory response by stimulating the synthesis ofplatelet activating factor (PAF) in endothelial cells [S. Prescottet.al., "Human Endothelial Cells in Culture Produce Platelet-ActivatingFactor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) When StimulatedWith Thrombin", Proc. Natl. Acad. Sci. USA, 81, pp. 3534-38 (1984)]. PAFis exposed on the surface of endothelial cells and serves as a ligandfor neutrophil adhesion and subsequent degranulation [G. M. Vercollettiet al., "Platelet-Activating Factor Primes Neutrophil Responses toAgonists: Role in Promoting Neutrophil-Mediated Endothelial Damage",Blood, 71, pp. 1100-07 (1988)]. Alternatively, thrombin may promoteinflammation by increasing vascular permeability which can lead to edema[P. J. Del Vecchio et al., "Endothelial Monolayer Permeability ToMacromolecules", Fed. Proc., 46, pp. 2511-15 (1987)]. Reagents whichblock the active site of thrombin, such as hirudin, interrupt theactivation of platelets and endothelial cells [C. L. Knupp, "Effect ofThrombin Inhibitors on Thrombin-Induced Release and Aggregation",Thrombosis Res., 49, pp 23-36 (1988)].

Thrombin has also been implicated in promoting cancer, based on theability of its native digestion product, fibrin, to serve as a substratefor tumor growth [A. Falanga et al., "Isolation and Characterization ofCancer Procoagulant: A Cysteine Proteinase from Malignant Tissue",Biochemistry, 24, pp. 5558-67 (1985); S. G. Gordon et al., "CysteineProteinase Procoagulant From Amnion-Chorion", Blood, 66, pp. 1261-65(1985); and A. Falanga et al., "A New Procoagulant In Acute Leukemia",Blood 71 pp 870-75 (1988)]. And thrombin has been implicated inneurodegenerative diseases based on its ability to cause neuriteretraction [D. Gurwitz et al., "Thrombin Modulates and ReversesNeuroblastoma Neurite Outgrowth", Proc. Natl. Acad. Sci. USA, 85, pp3440-44 (1988)]. Therefore, the ability to regulate the in vivo activityof thrombin has many important clinical implications.

Despite the developments to date, the need still exists for a moleculethat effectively inhibits thrombin function in clot formation, plateletactivation and various other thrombin-mediated processes and which canbe produced inexpensively and in commercially feasible quantities.

SUMMARY OF THE INVENTION

The present invention solves the problems enumerated above by providingmolecules which mimic the action of hirudin by binding to both the lowaffinity anion-binding exosite (ABE) and the catalytic site ofα-thrombin. These molecules are more potent than hirudin and, therefore,they may be administered to patients in dosages which are comparativelylower than those required in hirudin-based therapy regimens. Themolecules of this invention may be utilized in compositions and methodsfor inhibiting any thrombin-mediated or thrombin-associated function orprocess. Pharmaceutical compositions containing these molecules, as wellas methods of treatment or prophylaxis of vascular diseases,inflammatory responses, carcinomas, and neurodegenerative diseases usingthem are also part of the present invention. These molecules may also beemployed in compositions and methods for ex vivo imaging, for storingand treating extracorporeal blood and for coating invasive devices. Andthe molecules of this invention may be administered to a patient in acombination with a fibrinolytic agent to increase the efficacy of agiven dose of that agent or to lower the dose of that agent required fora given effect, such as dissolving a blood clot.

Due to their high potency and the fact that they may be prepared bychemical synthesis techniques, the molecules of the present inventionmay be prepared inexpensively, in commercially feasible amounts.Moreover, because the molecules of the present invention aresignificantly smaller than hirudin, they are less likely to stimulate anundesirable immune response in patients treated with them. Accordingly,the use of these thrombin inhibitors is not limited to the treatment ofacute disease. These molecules may also be utilized in therapy forchronic thromboembolic diseases, such atherosclerosis and restenosisfollowing angioplasty. The molecules of the present invention may alsobe utilized in a variety of other applications in place of natural orrecombinant hirudin.

As will be appreciated from the disclosure to follow, the molecules,compositions and methods of this invention are useful in the treatmentand prevention of various diseases attributed to the undesirable effectsof thrombin, as well as for diagnostic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an autoradiograph of an SDS-polyacrylamide geldemonstrating the binding of DNFB-[³⁵ S]-Sulfo-Try₆₃ hirudin₅₄₋₆₄ tohuman α-thrombin in the presence or absence of Sulfo-Tyr₆₃-N-acetylhirudin₅₃₋₆₄.

FIG. 2 depicts a three-dimensional model of human α-thrombin.

FIG. 3, panel A, depicts the effects of Hirulog-8 and Sulfo-Tyr₆₃hirudin₅₃₋₆₄ on the cleavage of Spectrozyme TH by human α-thrombin.

FIG. 3, panel B, depicts a Lineweaver-Burke plot of the cleavage ofSpectrozyme TH by human α-thrombin in the presence or absence of eitherHirulog-8 or Sulfo-Tyr₆₃ hirudin₅₃₋₆₄.

FIG. 4 depicts the effect of varying concentrations of Hirulog-8,hirudin, or Sulfo-Tyr63-N-acetyl-hirudin₅₃₋₆₄ on the activated partialthromboplastin time of normal human serum.

FIG. 5, panel A, depicts the time course for cleavage of varyingconcentrations of Hirulog-8 by human α-thrombin.

FIG. 5, panel B, depicts the relationship between Hirulog-8concentration and the duration of inhibition of Spectrozyme THhydrolysis by human α-thrombin.

FIG. 6 depicts the effect of linker length of the thrombin inhibitors ofthis invention on the inhibition of thrombin-catalyzed hydrolysis ofSpectrozyme TH.

FIG. 7 depicts the inhibitory effects of varying concentrations ofHirulog-8 or Sulfo-Tyr₆₃ -N-acetyl-hirudin₅₃₋₆₄ on the modification ofthrombin by ¹⁴ C-DFP.

FIG. 8 depicts the in vivo effect of varying doses of Hirulog-8 on APTTin baboons.

FIG. 9 depicts the comparative inhibitory effects of Hirulog-8 orheparin on the hydrolysis of fibrinogen by soluble or clot-boundthrombin.

FIG. 10 depicts the in vivo effects of varying doses of Hirulog-8 onplatelet deposition on an endarterectomized segment of baboon aorta.

FIG. 11 depicts the in vivo effects of varying doses of Hirulog-8 onplatelet deposition on a segment of collagen-coated tubing inserted intoa baboon.

FIG. 12 depicts the comparative in vivo effects of heparin, hirudin andHirulog-8 on platelet deposition on a segment of collagen-coated tubinginserted into a baboon AV shunt.

FIG. 13 depicts the in vivo effects of varying doses of Hirulog-8 onfibrin deposition on a segment of collagen-coated tubing inserted into ababoon AV shunt.

FIG. 14 depicts the change in APTT over time following intravenous bolusinjection of baboons with Hirulog-8.

FIG. 15 depicts the change in APTT over time following subcutaneousinjection of baboons with Hirulog-8.

FIG. 16 depicts the comparative in vivo effects of tissue plasminogenactivator together with either saline, heparin, hirudin or Hirulog-8 onreperfusion time in a rat model.

FIG. 17 depicts the comparative in vivo effects of tissue plasminogenactivator together with either saline, heparin, hirudin or Hirulog-8 onreocclusion time in a rat model.

FIG. 18 depicts the comparative in vivo effects of tissue plasminogenactivator together with either saline, heparin, hirudin or Hirulog-8 onAPTT in a rat model.

FIG. 19 depicts the comparative in vivo effects of tissue plasminogenactivator together with either saline, heparin, hirudin or Hirulog-8 onvessel patency in a rat model.

FIG. 20 depicts the effect of varying doses of Hirulog-8 on bleedingtimes in a baboon model.

DETAILED DESCRIPTION OF THE INVENTION

The following common abbreviations of the amino acids are usedthroughout the specification and in the claims:

    ______________________________________                                        Orn -- ornithine Gly -- glycine                                               Ala -- alanine   Val -- valine                                                Leu -- leucine   Ile -- isoleucine                                            Pro -- proline   Phe -- phenylalanine                                         Trp -- tryptophan                                                                              Met -- methionine                                            Ser -- serine    Thr -- threonine                                             Cys -- cysteine  Tyr -- tyrosine                                              Asn -- asparagine                                                                              Gln -- glutamine                                             Asp -- aspartic acid                                                                           Glu -- glutamic acid                                         Lys -- lysine    Arg -- arginine                                              His -- histidine Nle -- norleucine                                            Hyp -- hydroxyproline                                                                          Pgl -- phenylglycine                                         Ac -- acetyl     Suc -- succinyl                                              BOC -- tertButoxycarbonyl                                                                      Tos -- paraToluenesulfonyl                                   Cbz -- Carbobenzyloxy                                                                          D-Ala -- D-alanine                                           3,4,-dehydroPro -- 3,4,-                                                                       Sar -- sarcosine                                             dehydroproline   (N-methylglycine)                                            Tyr(OSO.sub.3 H) -- tyrosine                                                                   Tyr(SO.sub.3 H) -- tyrosine                                  sulfate          sulfonate                                                    3-,5-diiodoTyr -- 3-,5-                                                       diiodotyrosine                                                                ______________________________________                                    

The term "any amino acid" as used herein includes the L-isomers of thenaturally occurring amino acids, as well as other "non-protein" α-aminoacids commonly utilized by those in the peptide chemistry arts whenpreparing synthetic analogs of naturally occurring amino peptides. Thenaturally occurring amino acids are glycine, alanine, valine, leucine,isoleucine, serine, methionine, threonine, phenylalanine, tyrosine,tryptophan, cysteine, proline, histidine, aspartic acid, asparagine,glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithineand lysine. Examples of "non-protein" α-amino acids include norleucine,norvaline, alloisoleucine, homoarginine, thiaproline, dehydroproline,hydroxyproline (Hyp), homoserine, cyclohexylglycine(Chg)-amino-n-butyric acid (Aba), cyclohexylalanine (Cha),aminophenylbutyric acid (Pba), phenylalanines substituted at the ortho,meta, or para position of the phenyl moiety with one or two of thefollowing: a (C₁ -C₄) alkyl, a (C₁ -C₄) alkoxy, halogen or nitro groupsor substituted with a methylenedioxy group; β-2- and3-thienylal-alanine, β-2- and 3-furanylalanine, β-2-, 3- and4-pyridylalanine, β-(benzothienyl-2- and 3yl)alanine, β-(1- and2-naphthyl)alanine, O-alkylated derivatives of serine, threonine ortyrosine, S-alkylated cysteine, S-alkylated homocysteine, O-sulfate,O-phosphate and O-carboxylate esters of tyrosine, 3- and 5-sulfonyltyrosine, 3- and 5-carbonyl tyrosine, 3- and 5-phosphonyl tyrosine,4-methylsulfonyl tyrosine, 4-methylphosphonyl tyrosine, 4-phenylaceticacid, 3,5-diiodotyrosine, 3- and 5-nitrotyrosine, ε-alkyl lysine,delta-alkyl ornithine, and the D-isomers of the naturally occurringamino acids.

The compounds referred to herein as tyrosine sulfate, Tyr(OSO₃ H) andO-sulfate ester of tyrosine are identical and have the structuralformula: ##STR1##

The compounds referred to herein as tyrosine sulfonate, Tyr(SO₃ H),3-sulfonyl tyrosine and 5-sulfonyl tyrosine are identical and have thestructural formula: ##STR2##

The term "patient" as used in this application refers to any mammal,especially humans.

The term "anionic amino acid" as used herein means a meta, para orortho, mono- or di-substituted phenylalanine, cyclohexylalanine ortyrosine containing a carboxyl, phosphoryl or sulfonyl moiety, as wellas S-alkylated cysteine, S-alkylated homocysteine, γ-carboxyglutamicacid, ε-alkyl lysine, delta-alkyl ornithine, glutamic acid, and asparticacid. Examples of anionic amino acids are phosphothreonine,phosphoserine, phosphotyrosine, 3-, 4-, or 5-sulfotyrosine, 3-methylphosphonyltyrosine and 3-methyl sulfonyltyrosine.

The terms "catalytic site" "active site" and "active site pocket" asused herein, each refer to any or all of the following sites inthrombin: the substrate binding or "S₁ " site; the hydrophobic bindingor "oily" site; and the site where cleavage of a substrate is actuallycarried out ("charge relay site").

The term "N^(orn) " as used herein, refers to the side chain nitrogen ofornithine. The term "N⁹ " refers to any of the side chain nitrogens ofarginine. The term "N.sup.α " refers to the α-amino group of an aminoacid. And the term "pSi" as used in the specification and claims, refersto the replacement of an amide bond with the atoms designated inbrackets, according to the nomenclature described in J. Rudinger, InDrug Design, Vol. II, E. J. Ariens, ed., Academic Press, New York, p.319 (1971).

The term "backbone chain" as used herein, refers to the portion of achemical structure that defines the smallest number of consecutive bondsthat can be traced from one end of that chemical structure to the other.The atomic components that make up a backbone chain may comprise anyatoms that are capable of forming bonds with at least two other atoms.

For example, each of the following chemical structures is characterizedby a backbone chain of 7 atoms (the atoms which comprise the backbonechain are indicated in boldface): ##STR3##

The term "calculated length" as used in this application, refers to apredicted measurement derived by summing up the bond lengths between theatoms which comprise the backbone chain. Bond lengths between any twogiven atoms are well known in the art [see, for example, CRC Handbook ofchemistry and Physics, 65th Edition, R. C. Weist, ed., CRC Press, Inc.,Boca Raton, FL, pp. F-166-70 (1984)].

The present invention relates to molecules which bind to and inhibitthrombin. These molecules are characterized by three domains: acatalytic site-directed moiety ("CSDM"), a linker region, and an anionbinding exosite associating moiety ("ABEAM").

According to the present invention, the first domain, CSDM, binds to thecatalytic site of thrombin located at or near about Ser-195 and inhibitsor retards the amidolytic or estereolytic activity of thrombin.Preferably, CSDMs of the present invention are selected from one ofthree general groups: those which bind reversibly to thrombin and areslowly cleaved; those which bind reversibly to thrombin and cannot becleaved; and those which bind irreversibly to thrombin. Reversibleinhibitors bind to the active site of thrombin through non-covalentinteractions, such as ionic bonds, hydrophobic interactions or hydrogenbonding. Irreversible CSDMs form covalent bonds with thrombin.

According to a preferred embodiment, the CSDM which binds reversibly tothrombin and is slowly cleaved has the formula:

    X--A.sub.1 --A.sub.2 --A.sub.3 --Y,

wherein X is hydrogen or is characterized by a backbone chain consistingof from 1 to 35 atoms; A₁ is Arg, Lys or Orn; A₂ is a non-amide bond; A₃is characterized by a backbone chain consisting of from 1 to 9 atoms;and Y is a bond.

The non-amide bond component according to this embodiment may be formedby chemically modifying an amide bond. This may be achieved by methodswell known in the art [M. Szelke et al., "Potent New Inhibitors of HumanRenin", Nature, 299, pp. 555-57 (1982); D. H. Coy et al., "Facile SolidPhase Preparation of Proteins Containing the CH₂ --NH Peptide BondIsostere and Application to the Synthesis of Somatostatin (SRIF)Octapeptide Analogues", Peptides 1986, D. Theodoropoulos, Ed., WalterDeGruyter & Co., Berlin, pp. 143-46 (1987)]. When a non-amide bond isformed in this manner, it is preferable that the chemical modificationbe performed prior to the addition of the dipeptide containing this bondto the other components of CSDM or to the rest of the thrombin inhibitormolecule. In this manner, the dipeptide A₁ -A₂ --A₃ is added en bloc, ina single synthesis step, to the rest of the molecule.

According to a more preferred embodiment, A₁ is Arg and A₃ is Pro, D-Proor Sar. In this embodiment A₂ is a naturally occurring imide bond, whichis slowly cleaved by thrombin. This avoids having the necessity ofpre-forming the non-amide bond and allows A₁ and A₃ to be added to therest of the molecule sequentially rather than en bloc.

As set forth above, CSDMs according to this invention may bindirreversibly to thrombin. Examples of irreversible CSDMs include, butare not limited to, general serine proteinase inhibitors, such asphenylmethylsulfonylfluoride (PMSF), diisopropylfluorophosphate (DFP),tosylprolylchloromethylketone (TPCK) and tosyllysylchloromethylketone(TLCK); heterocyclic protease inhibitors, such as isocoumarins;thrombin-specific inhibitors, such as D--Phe--Pro--Arg--CHCl₂ (PPACK);and transition state analogues, such as difluoroketomethylene.

According to another preferred embodiment of the present invention,non-cleavable, reversible CSDMs consist of the formula:

    X--C.sub.1 --C.sub.2 --A.sub.3 -Y,

wherein C₁ is a derivative of Arg, Lys or Orn characterized by a reducedcarboxylate moiety or a carboxylate moiety that is displaced from theα-carbon by a chemical structure characterized by a backbone chain offrom 1 to 10 atoms; C₂ is a non-cleavable bond; and X, Y and A₃ are asdefined previously. Examples of C, components are β-homoarginine;arginine containing a reduced carboxylate moiety, such as Arg[psiCH₂NH]; β-homolysine and β-homoornithine.

Other non-cleavable, reversible CSDMs that may be employed in thethrombin inhibitors of this invention are benzamidine, DAPA, NAPAP andargatroban (argipidine).

For those thrombin inhibitors of this invention which have CSDM regionscharacterized by an A₂ or C₂ bond, the term "P₁ --P₁ '" sequence as usedherein, refers to the two chemical structures joined by said bond.

The X component of CSDM, which does not participate in actually bindingto the catalytic site, can be of unlimited length and variable make-up.However, for practical purposes and reduced cost of synthesis, X ispreferably characterized by a backbone chain consisting of from 1 to 35atoms and does not exceed a calculated length of 36Å. It is preferredthat X be a peptide, most preferably, D-Phe-Pro. This most preferableembodiment allows the X component to fit into a groove in thrombin thatis adjacent to the active site [S. Bajusz et al., "Inhibition ofThrombin and Trypsin by Tripeptide Aldehydes", Int. J. Peptide ProteinRes., 12, pp. 217-21 (1978); C. Kettner et al., "D--Phe--Pro--Arg--CH₂Cl--A Selective Affinity Label for Thrombin", Thromb. Res 14 pp 969-73(1979)]. This allows the CSDM component and therefore the molecules ofthe present invention, to bind to thrombin with an advantageously highdegree of affinity and optimal specificity.

According to the present invention, the second component of the thrombininhibitors of this invention is a linker region. Because the role ofthis portion of the molecule is to provide a bridge between the CSDM andthe ABEAM, it is the length of the linker, rather than its structure,that is of prime importance. The calculated length of the backbone chainwhich characterizes the linker must be at least about 18 Å--the distancebetween the catalytic site and the anion binding exosite ofthrombin--and less than about 42 Å.

The backbone chain of the linker may comprise any atoms which arecapable of bonding to at least two other atoms. Preferably, the backbonechain consists of any chemically feasible combination of atoms selectedfrom oxygen, carbon, nitrogen and sulfur. Those of skill in the art areaware of what combination of the above backbone chain atoms falls withinthe required length based on known distances between various bonds [see,for example, R. T. Morrison and R. N. Boyd, Organic Chemistry, 3rdEdition, Allyn and Bacon, Inc., Boston, Mass. (1977)]. According to apreferred embodiment, the linker is a peptide which comprises the aminoacid sequence Gly--Gly--Gly--Asn--Gly--Asp--Phe. Preferably, the aminoacid bound to the ABEAM component is Phe.

The third domain of the thrombin inhibitors of this invention is theABEAM which binds to the anion binding exosite of thrombin. Preferablythe ABEAM has the formula:

    W--B.sub.1 --B.sub.2 --B.sub.3 -B.sub.4 --B.sub.5 --B.sub.6 --B.sub.7 --B.sub.8 --Z;

wherein W is a bond; B₁ is an anionic amino acid; B₂ is any amino acid;B₃ is Ile, Val, Leu, Nle or Phe; B₄ is Pro, Hyp, 3,4-dehydroPro,thiazolidine-4-carboxylate, Sar, any N-methyl amino acid or D--Ala; B₅is an anionic amino acid; B₆ is an anionic amino acid; B₇ is alipophilic amino acid selected from the group consisting of Tyr, Trp,Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of one ofthese lipophilic amino acids and any amino acid; B₈ is a bond or apeptide containing from one to five residues of any amino acid; and Z isa carboxy terminal residue selected from OH, C₁ -C₆ alkoxy, amino, mono-or di-(C₁ -C₄) alkyl substituted amino or benzylamino.

Peptides which are homologous to the carboxy terminal portion of hirudinhave been shown to bind to the anion binding exosite on thrombin[copending United States patent application 314,756 and J. M. Maraganoreet al., "Anticoagulant Activity of Synthetic Hirudin Peptides", J. Biol.Chem., 264, pp. 8692-98 (1989); both of which are herein incorporated byreference].

According to a preferred embodiment of this invention, ABEAM ishomologous to amino acids 56-64 of hirudin, i. e., B₁ is Glu; B₂ is Glu;B₃ is Ile; B₄ is Pro; B₅ is Glu; B₆ is Glu; B₇ is Tyr-Leu, Tyr (SO₃H)-Leu or Tyr(OSO₃ H)-Leu, or (3-,5-diiodoTyr)-Leu; B₈ is a bond; and Zis OH. It should be noted that native hirudin contains Tyr(OSO₃ H) atposition 63. However, carboxy terminal hirudin peptides which containTyr(SO₃ H) have identical anticoagulant activity as those which containthe native Tyr(OSO₃ H) [see copending United States patent application314,756].

Other ABEAM components within the scope of this invention may comprisethose portions of any molecule known to bind to the anion binding siteof thrombin. These include amino acids 1675-1686 of Factor V, aminoacids 272-285 of platelet glycoproten Ib, amino acids 415-428 ofthrombomodulin, amino acids 245-259 of prothrombin Fragment 2 and aminoacids 30 to 44 of fibrinogen Aα chain. In addition, the ABEAM componentmay be selected from any of the hirudin peptide analogues described byJ. L. Krstenansky et al., "Development of MDL-28,050, A Small StableAntithrombin Agent Based On A Functional Domain of the Leech Protein,Hirudin", Thromb Haemostas., 63, pp 208-14 (1990).

The preferred thrombin inhibitors of this invention are termed Hirulogs,and are described in the subsequent examples. The most preferredHirulogs are Hirulog-8, Hirulog-12, Hirulog-18a, Hirulog-18b andHirulog-33. Hirulog-8, -12 and -33 are reversible thrombin inhibitorsthat are slowly cleaved. Hirulog-18a and -18b are reversible inhibitorswhich are not cleaved.

The thrombin inhibitors of the present invention may be synthesized byvarious techniques which are well known in the art. These includeenzymatic cleavage of natural or recombinant hirudin, recombinant DNAtechniques, solid-phase peptide synthesis, solution-phase peptidesynthesis, organic chemical synthesis techniques, or a combination ofthese techniques. The choice of synthesis technique will, of course,depend upon the composition of the particular inhibitor. In a preferredembodiment of this invention, the thrombin inhibitor is entirelypeptidic and is synthesized by solid-phase peptide synthesis techniques,solution-phase peptide synthesis techniques or a combination thereofwhich constitute the most cost-efficient procedures for producingcommercial quantities of these molecules.

When "non-protein" amino acids are contained in the thrombin inhibitormolecule, they may be either added directly to the growing chain duringpeptide synthesis or prepared by chemical modification of the completesynthesized peptide, depending on the nature of the desired"non-protein" amino acid. Those of skill in the chemical synthesis artare well aware of which "non-protein" amino acids may be added directlyand which must be synthesized by chemically modifying the completepeptide chain following peptide synthesis.

The synthesis of those thrombin inhibitors of this invention whichcontain both non-amino acid and peptidic portions is preferably achievedby a mixed heterologous/solid phase technique. This technique involvesthe solid-phase synthesis of all or most of the peptide portion of themolecule, followed by the addition of the non-amino acid componentswhich are synthesized by solution phase techniques. The non-amino acidmay be coupled to the peptidic portion via solid-phase or solution-phasemethods. Similarly, any remaining peptidic portions may also be addedvia solid-phase or solution phase methods.

The molecules of the present invention display potent anticoagulantactivity. This activity may be assayed in vitro using any conventionaltechnique. Preferably, an assay for anticoagulant activity involvesdirect determination of the thrombin-inhibitory activity of themolecule. Such techniques measure the inhibition of thrombin-catalyzedcleavage of colorimetric substrates or, more preferably, the increase inthrombin times or increase in activated partial thromboplastin times ofhuman plasma. The latter assay measures factors in the "intrinsic"pathway of coagulation. Alternatively, the assay employed may usepurified thrombin and fibrinogen to measure the inhibition of release offibrinopeptides A or B by radioimmunoassay or ELISA.

The antiplatelet activity of the molecules of this invention may also bemeasured by any of a number of conventional platelet assays. Preferably,the assay will measure a change in the degree of aggregation ofplatelets or a change in the release of a platelet secretory componentin the presence of thrombin. The former may be measured in anaggregometer. The latter may be measured using RIA or ELISA techniquesspecific for the secreted component.

The molecules of the present invention are useful in compositions,combinations and methods for the treatment and prophylaxis of variousdiseases attributed to thrombin-mediated and thrombin-associatedfunctions and processes. These include myocardial infarction, stroke,pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion,restenosis following arterial injury or invasive cardiologicalprocedures, acute or chronic atherosclerosis, edema and inflammation,various cell regulatory processes (e.g. secretion, shape changes,proliferation), cancer and metastasis, and neurodegenerative diseases.

The thrombin inhibitors of the present invention may be formulated usingconventional methods to prepare pharmaceutically useful compositions,such as the addition of a pharmaceutically acceptable carrier. Thesecompositions and the methods employing them may be used for treating orpreventing thrombotic diseases in a patient.

According to an alternate embodiment of the present invention, thethrombin inhibitors may be employed in combinations, compositions, andmethods for treating thrombotic disease, and for decreasing the dosageof a thrombolytic agent required to establish reperfusion or preventreocclusion in a patient. Additionally, the thrombin inhibitors of thisinvention may be used in combinations, compositions, and methods fordecreasing reperfusion time or increasing reocclusion time in a patienttreated with a thrombolytic agent. These combinations and compositionscomprise a pharmaceutically effective amount of a thrombin inhibitor ofthe present invention and a pharmaceutically effective amount of athrombolytic agent.

In these combinations and compositions, the thrombin inhibitor and thethrombolytic agent work in a complementary fashion to dissolve bloodclots, resulting in decreased reperfusion times and increasedreocclusion times in patients treated with them. Specifically, thethrombolytic agent dissolves the clot, while the thrombin inhibitorprevents newly exposed, clot-entrapped or clot-bound thrombin fromregenerating the clot. The use of the thrombin inhibitor in thecombinations and compositions of this invention advantageously allowsthe administration of a thrombolytic reagent in dosages previouslyconsidered too low to result in thrombolytic effects if given alone.This avoids some of the undesirable side effects associated with the useof thrombolytic agents, such as bleeding complications.

Thrombolytic agents which may be employed in the combinations andcompositions of the present invention are those known in the art. Suchagents include, but are not limited to, tissue plasminogen activatorpurified from natural sources, recombinant tissue plasminogen activator,streptokinase, urokinase, prourokinase, anisolated streptokinaseplasminogen activator complex (ASPAC), animal salivary gland plasminogenactivators and known, biologically active derivatives of any of theabove.

The term "combination" as used herein, includes a single dosage formcontaining at least one thrombin inhibitor of this invention and atleast one thrombolytic agent; a multiple dosage form, wherein thethrombin inhibitor and the thrombolytic agent are administeredseparately, but concurrently; or a multiple dosage form wherein the twocomponents are administered separately, but sequentially. In sequentialadministration, the thrombin inhibitor may be given to the patientduring the time period ranging from about 5 hours prior to about 5 hoursafter administration of the thrombolytic agent. Preferably, the thrombininhibitor is administered to the patient during the period ranging from2 hours prior to 2 hours following administration of the thrombolyticagent.

Alternatively, the thrombin inhibitor and the thrombolytic agent may bein the form of a single, conjugated molecule. Conjugation of the twocomponents may be achieved by standard cross-linking techniques wellknown in the art. The single molecule may also take the form of arecombinant fusion protein, if both the thrombin inhibitor and thethrombolytic agent are peptidic.

Various dosage forms may be employed to administer the compositions andcombinations of this invention. These include, but are not limited to,parenteral administration, oral administration and topical application.The compositions and combinations of this invention may be administeredto the patient in any pharmaceutically acceptable dosage form, includingthose which may be administered to a patient intravenously as bolus orby continued infusion, intramuscularly--including paravertebrally andperiarticularly--subcutaneously, intracutaneously, intra-articularly,intrasynovially, intrathecally, intra-lesionally, periostally or byoral, nasal, or topical routes. Such compositions and combinations arepreferably adapted for topical, nasal, oral and parenteraladministration, but, most preferably, are formulated for parenteraladministration.

Parenteral compositions are most preferably administered intravenouslyeither in a bolus form or as a constant infusion. If the thrombininhibitor is being used as an antiplatelet compound, constant infusionis preferred. If the thrombin inhibitor is being used as ananticoagulant, a subcutaneous or intravenous bolus injection ispreferred. For parenteral administration, fluid unit dose forms areprepared which contain a thrombin inhibitor of the present invention anda sterile vehicle. The thrombin inhibitor may be either suspended ordissolved, depending on the nature of the vehicle and the nature of theparticular thrombin inhibitor. Parenteral compositions are normallyprepared by dissolving the thrombin inhibitor in a vehicle, optionallytogether with other components, and filter sterilizing before fillinginto a suitable vial or ampule and sealing. Preferably, adjuvants suchas a local anesthetic, preservatives and buffering agents are alsodissolved in the vehicle. The composition may then be frozen andlyophilized to enhance stability.

Parenteral suspensions are prepared in substantially the same manner,except that the active component is suspended rather than dissolved inthe vehicle. Sterilization of the compositions is preferably achieved byexposure to ethylene oxide before suspension in the sterile vehicle.Advantageously, a surfactant or wetting agent is included in thecomposition to facilitate uniform distribution of its components.

Tablets and capsules for oral administration may contain conventionalexcipients, such as binding agents, fillers, diluents, tableting agents,lubricants, disintegrants, and wetting agents. The tablet may be coatedaccording to methods well known in the art. Suitable fillers which maybe employed include cellulose, mannitol, lactose and other similaragents. Suitable disintegrants include, but are not limited to, starch,polyvinylpyrrolidone and starch derivatives, such as sodium starchglycolate. Suitable lubricants include, for example, magnesium stearate.Suitable wetting agents include sodium lauryl sulfate.

Oral liquid preparations may be in the form of aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives. These include suspending agents; such assorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose,carboxymethylcellulose, aluminum stearate gel or hydrogenated ediblefats; emulsifying agents which include lecithin, sorbitan monooleate,polyethylene glycols, or acacia; non-aqueous vehicles, such as almondoil, fractionated coconut oil, and oily esters; and preservatives, suchas methyl or propyl p-hydroxybenzoate or sorbic acid.

Compositions formulated for topical administration may, for example, bein aqueous jelly, oily suspension or emulsified ointment form.

The dosage and dose rate of the thrombin inhibitor will depend on avariety of factors, such as the size of the patient, the specificpharmaceutical composition used, the object of the treatment, i.e.,therapy or prophylaxis, the nature of the thrombotic disease to betreated, and the judgment of the treating physician.

According to the present invention, a preferred pharmaceuticallyeffective daily dose of the thrombin inhibitor of this invention isbetween about 1 μg/kg body weight of the patient to be treated ("bodyweight") and about 5 mg/kg body weight. In combinations containing athrombolytic agent, a pharmaceutically effective daily dose of thethrombolytic is between about 10% and 80% of the conventional dosagerange. The "conventional dosage range" of a thrombolytic agent is thedaily dosage used when that agent is employed in a monotherapy.[Physician's Desk Reference 1989, 43rd Edition, Edward R. Barnhart,publisher]. That conventional dosage range will, of course, varydepending on the thrombolytic agent employed. Examples of conventionaldosage ranges are as follows: urokinase--500,000 to 6,250,000units/patient; streptokinase--140,000 to 2,500,000 units/patient;tPA--0.5 to 5.0 mg/kg body weight; ASPAC--0.1 to 10 units/kg bodyweight.

Most preferably, the therapeutic and prophylactic compositions of thepresent invention comprise a dosage of between about 10 μg/kg bodyweight and about 500 μg/kg body weight of the thrombin inhibitor. Mostpreferred combinations comprise the same amount of the thrombininhibitor and between about 10% and about 70% of the conventional dosagerange of a thrombolytic agent. It should also be understood that a dailypharmaceutically effective dose of either the thrombin inhibitors ofthis invention or the thrombolytic agent present in combinations of theinvention, Nay be less than or greater than the specific ranges citedabove.

Once improvement in the patient's condition has occurred, a maintenancedose of a combination or composition of this invention is administered,if necessary. Subsequently, the dosage or the frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. When thesymptoms have been alleviated to the desired level, treatment shouldcease. Patients may, however, require intermittent treatment upon anyrecurrence of disease symptoms.

According to an alternate embodiment of this invention, thrombininhibitors may be used in compositions and methods for coating thesurfaces of invasive devices, resulting in a lower risk of clotformation or platelet activation in patients receiving such devices.Surfaces that may be coated with the compositions of this inventioninclude, for example, prostheses, artificial valves, vascular grafts,stents and catheters. Methods and compositions for coating these devicesare known to those of skill in the art. These include chemicalcross-linking or physical adsorption of the thrombininhibitor-containing compositions to the surfaces of the devices.

According to a further embodiment of the present invention, thrombininhibitors may be used for ex vivo thrombus imaging in a patient. Inthis embodiment, the thrombin inhibitor is labelled with a radioisotope.The choice of radioisotope is based upon a number of well-knownfactors., for example, toxicity, biological half-life and detectability.Preferred radioisotopes include, but are not limited to, ¹²⁵ I, ¹²³ Iand ¹¹¹ In. Techniques for labelling the thrombin inhibitor are wellknown in the art. Most preferably, the radioisotope is ¹²³ I and thelabelling is achieved using ¹²³ I-Bolton-Hunter Reagent. The-labelledthrombin inhibitor is administered to a patient and allowed to bind tothe thrombin contained in a clot. The clot is then observed by utilizingwell-known detecting means, such as a camera capable of detectingradioactivity coupled to a computer imaging system. This technique alsoyields images of platelet-bound thrombin and meizothrombin.

This invention also relates to compositions containing the thrombininhibitors of this invention and methods for using such compositions inthe treatment of tumor metastases. The efficacy of the thrombininhibitors of this invention for the treatment of tumor metastases ismanifested by the inhibition of metastatic growth. This is based uponthe presence of a procoagulant enzyme in certain cancer cells. Thisenzyme activates the conversion of Factor X to Factor Xa in thecoagulation cascade, resulting in fibrin deposition which, in turn,serves as a substrate for tumor growth. By inhibiting fibrin depositionthrough the inhibition of thrombin, the molecules of the presentinvention serve as effective anti-metastatic tumor agents. Examples ofmetastatic tumors which may be treated by the thrombin inhibitors ofthis invention include, but are not limited to, carcinoma of the brain,carcinoma of the liver, carcinoma of the lung, osteocarcinoma andneoplastic plasma cell carcinoma.

The invention also relates to methods and compositions employing theabove-described thrombin inhibitors to inhibit thrombin-inducedendothelial cell activation. This inhibition includes the repression ofplatelet activation factor (PAF) synthesis by endothelial cells. Thesecompositions and methods have important applications in the treatment ofdiseases characterized by thrombin-induced inflammation and edema, whichis thought to be mediated be PAF. Such diseases include, but are notlimited to, adult respiratory distress syndrome, septic shock,septicemia and reperfusion damage.

Early stages of septic shock include discrete, acute inflammatory andcoagulopathic responses. It has previously been shown that injection ofbaboons with a lethal dose of live E. coli leads to marked declines inneutrophil count, blood pressure and hematocrit. Changes in bloodpressure and hematocrit are due in part to the generation of adisseminated intravascular coagulopathy (DIC) and have been shown toparallel consumption of fibrinogen [F. B. Taylor et al., "Protein CPrevents the Coagulopathic and Lethal Effects of Escherichia coliinfusion in the Baboon", J. Clin. Invest., 79, pp. 918-25 (1987)].Neutropenia is due to the severe inflammatory response caused by septicshock which results in marked increases in tumor necrosis factor levels.The thrombin inhibitors of this invention may be utilized incompositions and methods for treating or preventing DIC in septicemiaand other diseases.

This invention also relates to the use of the above-described thrombininhibitors, or compositions comprising them, as anticoagulants forextracorporeal blood. As used herein, the term "extracorporeal blood"includes blood removed in line from a patient, subjected toextracorporeal treatment, and then returned to the patient in suchprocesses as dialysis procedures, blood filtration, or blood bypassduring surgery. The term also includes blood products which are storedextracorporeally for eventual administration to a patient and bloodcollected from a patient to be used for various assays. Such productsinclude whole blood, plasma, or any blood fraction in which inhibitionof coagulation is desired.

The amount or concentration of thrombin inhibitor in these types ofcompositions is based on the volume of blood to be treated or, morepreferably, its thrombin content. Preferably, an effective amount of athrombin inhibitor of this invention for preventing coagulation inextracorporeal blood is from about 1 μg/60 ml of extracorporeal blood toabout 5 mg/60 ml of extracorporeal blood.

The thrombin inhibitors of this invention may also be used to inhibitclot-bound thrombin, which is believed to contribute to clot accretion.This is particularly important because commonly used anti-thrombinagents, such as heparin and low molecular weight heparin, areineffective against clot-bound thrombin.

Finally, the thrombin inhibitors of this invention may be employed incompositions and methods for treating neurodegenerative diseases.Thrombin is known to cause neurite retraction, a process suggestive ofthe rounding in shape changes of brain cells and implicated inneurodegenerative diseases, such as Alzheimer's disease and Parkinson'sdisease.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLE 1 Synthesis of Sulfo-Try₆₃ hirudin₅₄₋₆₄

Sulfo-Tyr₆₃ hirudin₅₄₋₆₄ has the amino acid formula:H--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr(OSO₃)--Leu--OH. Weprepared this peptide by solid-phase peptide synthesis employing anApplied Biosystems 430 A Peptide Synthesizer (Applied Biosystems, FosterCity, Calif.).

Specifically, we reacted 0.259 meq of BOC--O--Leu resin (1% DVB resin)sequentially with 2 mmoles of protected amino acids. Following 10 cyclesof synthesis, we deprotected the peptide and uncoupled it from the DVBresin by treatment with anhydrous HF: p-cresol: ethyl methyl sulfate(10:1:1, v/v/v). The peptide was further purified on a Vydac C₁₈ HPLCreverse phase column (22 mm×25 cm) which had previously beenequilibrated in 0.1% TFA in water. Prior to applying the peptide to thecolumn, we dissolved it in 2.0 ml of 0.1% TFA in water. If necessary, anadditional 1 ml of 6M guanidinium chloride was added to the sample toincrease solubility. After we applied the sample, the column wasdeveloped with a linear gradient of increasing acetonitrile (0-80%) in0.1% TFA over 45 minutes at a flow rate of 4.0 ml/min. The effluentstream was monitored at 229 nm and fractions were collected manually.

We sulfated the resulting purified peptide at the single tyrosineresidue using standard methodology [T. Nakahara et al., "Preparation ofTyrosine-O-[³⁵ S] Sulfated Cholecystokinin Octapeptide From ANon-Sulfated Precursor Peptide", Anal. Biochem,, 154, pp. 194-99(1986)]. Sulfo-Tyr₆₃ hirudin₅₄₋₆₄ was then purified away from otherpeptides and reaction components by reverse-phase HPLC employing a VydacC₁₈ column (4.6×25 cm) and an Applied Biosystems liquid chromatographicsystem. The column was equilibrated in a 0.1% TFA/water solvent anddeveloped with a linear gradient of increasing acetonitrileconcentration from 0 to 35% over 90 minutes at a flow rate of 0.8 ml/minwith a 0.085% TFA-containing solvent. Fractions were assayed forabsorbance at 214 nm.

EXAMPLE 2 Crosslinking of Human Thrombin With Sulfo-TYr₆₃-Dinitrofluorobenzyl-hirudin₅₄₋₆₄

We prepared Sulfo-Tyr₆₃ -dinitrofluorobenzylhirudin₅₄₋₆₄ (Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄) by reacting Sulfo-Try₆₃ hirudin₅₄₋₆₄ (2.0 mg;prepared as in Example 1) with a stoichiometric quantity ofdifluorodinitrobenzene (Pierce Chemical Co., Rockford, Ill.) indimethylformamide (DMF) for 18 hours at room temperature. We thensubjected the sample to analytical HPLC separation employing an AppliedBiosystems 150 A Liquid Chromatographic System and a Brownlee RP-300 C,column (0.46×10 cm) to determine the extent of derivatization. Thecolumn was equilibrated in 0.1% TFA in water (solvent A) and developedwith a 0-50% linear gradient of 0.085% TFA/70% acetonitrile (solvent B)over 45 min and then 50-100% linear gradient of solvent B over 15 min.We used a constant flow rate of 1.0 ml/minute.

The effluent stream was monitored at 214 nm and 310 nm for absorbance.Peptide derivatized with the difluorodinitrobenzene reagent absorbs at310 nm. We found that the above-described reaction produced Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄ at 15-30% yield. Following synthesis, Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄ was stored in the same dimethylformamide solvent at-20° C. for up to 1 month.

We reacted a 10-fold molar excess of Sulfo-Tyr₆₃ -DNFB-hirudin₅₄₋₆₄ withhuman α-thrombin (12.5 mg) for 18 hr at room temperature in aphosphate-buffered saline. We determined the extent of cross-linking byanalyzing the reaction mixture on an SDS-polyacrylamide gel. SDS-PAGEshowed a decrease in the relative mobility of the α-thrombin bandreflective of an increase in molecular weight of 1000-2000 daltons (Da).This shift is consistent with cross-linking of thrombin with Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄ at a single site.

We confirmed that formation of a covalent complex between Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄ and human thrombin is specific by using [³⁵S]-Sulfo-Tyr₆₃ -DNFB-hirudin₅₄₋₆₄. [³⁵ S]-Sulfo-Tyr₆₃ -DNFB-hirudin₅₄₋₆₄was prepared essentially as described above using H₂ [³⁵ S]O₄ instead ofH₂ SO₄ in the Nakahara sulfation procedure [see also, copending UnitedStates patent applications Serial Nos. 164,178, 251,150, 280,618, and314,756, and J. M. Maraganore et al., "Anticoagulant Activity ofSynthetic Hirudin Peptides", J. Biol. Chem., 264, pp. 8692-98 (1989) allof which are herein incorporated by reference].

We reacted [³⁵ S]-Sulfo-Tyr₆₃ -DNFB-hirudin₅₄₋₆₄ with human α-thrombin,either in the presence or absence of a 5- or 20-fold molar excess (overthe concentration of thrombin) of Sulfo-Tyr₆₃ -N-acetylhirudin₆₃₋₆₄(prepared as in Example 1 with the addition of N-acetyl asparagine as afinal step in peptide synthesis). Following incubation at roomtemperature for 18 hrs, we subjected the mixture to SDS-PAGE andautoradiography. The results (FIG. 1) showed that [³⁵ S]-labeled peptidewas incorporated into the band which represents thrombin and that thepresence of cold, unlabeled hirudin peptide attenuated the 35 magnitudeof covalent complex formation to <10%. Thus, reaction of Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄ with thrombin results in the 1:1 stoichiometricbinding of the hirudin peptide at a specific binding site.

In order to identify the site on thrombin where Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄ binds, thrombin/Sulfo-Tyr₆₃-dinitrobenzyl(DNB)-hirudin₅₄₋₆₄ complex (1.0 mg) was applied to aSephadex G-50 column (1.5×45 cm) which was equilibrated and developedwith 7M urea, 20 mM Tris, pH 7.5. This chromatography removed anyunreacted Sulfo-Tyr₆₃ -DNFB-hirudin₆₄₋₆₄. A peak containingthrombin/Sulfo-Tyr₆₃ -DNB-hirudin₅₄₋₆₄ was isolated in the void volumefractions, pooled and reduced by the addition of 10 μl ofβ-mercaptoethanol.

Following reduction, we S-carboxymethylated the complex using iodoaceticacid as previously described [J. M. Maraganore et al., "A New Class ofPhospholipases A₂ with Lysine in Place of Aspartate-49", J. Biol. Chem,259, pp. 13839-43 (1984)]. The reduced, S-alkylated protein was thendialyzed extensively against 3% acetic acid at room temperature.Following dialysis, we digested the complex with pepsin (2% w/v) for 4hr at 37° C. Peptic fragments of reduced, S-carboxymethylatedthrombin/Sulfo-Tyr₆₃ -DNB-hirudin₅₄₋₆₄ were purified by reverse-phaseHPLC using an Aquapore RP-300 C₈ column (0.46×10 cm). The column wasequilibrated in 0.1% TFA in water and developed with a gradient ofincreasing 0.085% TFA/70% acetonitrile (0-60%) over 80 minutes at a flowrate of 1.0 ml/min. The effluent stream was monitored for absorbance atboth 214 and 310 nm. Fractions of 1.0 ml were collected automatically.HPLC separation of peptic fragments allowed resolution of a single majorpeak of both 214 and 310 nm-absorbing material. Because of its far UVabsorbance, this fragment contained the bound Sulfo-Tyr₆₃-DNFB-hirudin₅₄₋₆₄.

We then subjected the fragment to automated Edman degradation with anApplied Biosystems 470A gas-phase sequencer equipped with a 900A datasystem. Phenylthiohydantoin (PTH) amino acids were analyzed online usingan Applied Biosystems 120A PTH analyzer and a PTH-C₁₈ column (2.1×220mm). Shown below is a table of repetitive yields from the sequenceanalysis:

    ______________________________________                                        Cycle         Amino Acid pmoles                                               ______________________________________                                         1            Lys        858.5                                                 2            Glu        629.2                                                 3            Thr        357.6                                                 4            Trp        276.3                                                 5            Thr        289.0                                                 6            Ala        474.4                                                 7            Asn        369.0                                                 8            Val        490.7                                                 9            Gly        296.1                                                10            (x)        (--)                                                 11            Gly        267.2                                                12            Gln        208.8                                                13            Pro        103.5                                                14            Ser         21.6                                                15            Val         23.3                                                ______________________________________                                    

The peptide sequence was found to correspond to residues 144-154 ofhuman α-thrombin [J. W. Fenton, II., "Thrombin Active Site Regions"Semin. Thromb. Hemostasis, 12, pp. 200-08 (1986)]. Peptic cleavagesoccurred at a Leu-Lys and Val-Leu bond, consistent with the specificityof this enzyme.

In the course of sequence analysis, the amino acid corresponding toLys-149 (cycle 10) could not be identified or quantitated. This probablyresulted from derivatization of the ε-NH₂ group of this amino acid withthe dinitrofluorobenzyl moiety of Sulfo-Tyr₆₃ -DNFB-hirudin₅₄₋₆₄. Thus,Lys-149 is the major site where Sulfo-Tyr₆₃ -DNFB-hirudin₅₄₋₆₄ reactswith α-thrombin.

EXAMPLE 3 Design of a Thrombin Inhibitor Capable of Blocking theCatalytic Site and Binding to the Anion Binding Exosite

Carboxy terminal hirudin peptides effectively block thrombin-catalyzedfibrinogen hydrolysis, but not chromogenic substrate hydrolysis [J. M.Maraganore et al., J. Biol. Chem., 264, pp. 8692-98 (1989)]. Inaddition, hirudin peptides do not neutralize thrombin-catalyzedactivation of Factors V and VIII [J. W. Fenton, II, et al , "HirudinInhibition by Thrombin", Anqio. Archiv. Biol., 18, p. 27 (1989)].

Hirudin peptides, such as Sulfo-Tyr₆₃ -N-acetyl-hirudin₅₃₋₆₄, exhibitpotent inhibitory effects toward thrombin-induced platelet activation invitro [J. A. Jakubowsky and J. M. Maraganore, "Inhibition ofThrombin-Induced Platelet Activities By A Synthetic 12 Amino AcidResidue Sulfated Peptide (Hirugen)" Blood, p. 1213 (1989)].Nevertheless, a thrombin inhibitor capable of blocking the active sitemay be required for inhibition of platelet thrombosis in vivo, ifactivation of Factors V and VIII are rate-limiting steps. Thisconclusion is warranted from results obtained with the irreversiblethrombin inhibitor (D--Phe)--Pro--Arg--CH₂ Cl [S. R. Hanson and L. A.Harker, "Interruption of Acute Platelet-Dependent Thrombosis by theSynthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginyl ChloromethylKetone", Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)] and otherreversible thrombin inhibitors [J. F. Eidt et al., "Thrombin is anImportant Mediator of Platelet Aggregation in Stenosed Canine CoronaryArteries with Endothelial Injury", J. Clin. Invest., 84, pp. 18-27(1989)].

Using the above knowledge that the NH₂ -terminus of hirudin peptides isproximal to Lys-149, we employed a three-dimensional model of thrombin(FIG. 2) [B. Furie, et al., "Computer-Generated Models of BloodCoagulation Factor Xa, Factor IXa, and Thrombin Based Upon StructuralHomology with Other Serine Proteases", J. Biol. Chem., 257, pp. 3875-82(1982)] to design an agent which: 1) binds to the anion binding exositeof thrombin; and, 2) is capable of blocking the active site pocket ofthrombin and inhibiting the function of catalytic residues containedtherein.

We determined that the minimal distance from the ε-NH₂ of Lys-149 to theβ-hydroxylate of Ser-195 is 18-20 Å. Based on a 3 Å/amino acid residuelength, we calculated that at least about 4-7 amino acids would berequired to link a hirudin peptide, such as Sulfo-Tyr₆₃ hirudin₅₃₋₆₄, toa domain comprising an active-site inhibitor structure. The compositionof the linker was designed to be glycine. Glycine was chosen in order toengineer the greatest flexibility of a linker for these preliminaryinvestigations. It should be understood, however, that other, more rigidbiopolymer linkers may also be employed.

We chose the sequence (D--Phe)--Pro--Arg--Pro as the active siteinhibitor because thrombin exhibits specificity for Arg as the P₁ aminoacid in the cleavage of substrates. A Pro following the Arg yields abond that is cleaved very slowly by thrombin. We designed alternatepeptides by replacing Pro (following the P₁ Arg) with a sarcosyl- orN-methyl-alanine amino acid or by chemical reduction of an Arg--Glyscissile bond.

EXAMPLE 4 Synthesis of Hirulog-8

Hirulog-8 has the formula: H-(D--Phe)--Pro--Arg--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. Wesynthesized Hirulog-8 by conventional solid-phase peptide synthesisemploying an Applied Biosystems 430 A Peptide Synthesizer. This peptidewas synthesized using BOC-L-Leucine-O-divinylbenzene resin. Additionalt-BOC-amino acids (Peninsula Laboratories, Belmont, Calif.) usedincluded BOC-O-2,6-dichlorobenzyl tyrosine, BOC-L-glutamic acid(γ-benzyl ester), BOC-L-proline, BOC-L-isoleucine, BOC-L-phenylalanine,BOC-L-aspartic acid (β-benzyl ester), BOC-glycine, BOC-L-asparagine,BOC-D-phenylalanine, and BOC-L-arginine. In order to achieve higheryields in synthesis, the (Gly)₄ linker segment was attached in twocycles of manual addition of BOC-glycylglycine (Beckman Biosciences,Inc., Philadelphia, Pa.). After completion of synthesis, the peptide wasfully deprotected and uncoupled from the divinylbenzene resin bytreatment with anhydrous HF: p-cresol: ethylmethyl sulfate (10:1:1,v/v/v). Following removal from the resin, the peptide was lyophilized todryness.

Crude Hirulog-8 was purified by reverse-phase HPLC employing an AppliedBiosystems 151A liquid chromatographic system and a Vydac C₁₈ column(2.2×25 cm). The column was equilibrated in 0.1% TFA/water and developedwith a linear gradient of increasing acetonitrile concentration from 0to 80% over 45 minutes in the 0.1% TFA at a flow-rate of 4.0 ml/min. Theeffluent stream was monitored for absorbance at 229 nm and fractionswere collected manually. We purified 25-30 mg of crude Hirulog-8 by HPLCand recovered 15-20 mg of pure peptide.

We confirmed the structure of purified Hirulog-8 by amino acid andsequence-analyses. Amino acid hydrolysates were prepared by treating thepeptide with 6N HCl, in vacuo, at 110° C. for 24 hrs. We then analyzedthe hydrolysates by ion-exchange chromatography and subsequent ninhydrinderivatization/detection using a Beckman 6300 automated analyzer. Weperformed sequence analysis using automated Edman degradation on anApplied Biosystems 470A gas-phase 10 sequencer equipped with a Model900A data system. Phenylthiohydantoin (PTH) amino acids were analyzedon-line using an Applied Biosystems 120A PTH-analyzer and a PTH-C,,column (2.1×220 mm).

EXAMPLE 5 Synthesis Of Hirulog-9

Hirulog-9 has the formula: H--(D--Phe)--Pro--Arg--D--Pro-(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. Wesynthesized this peptide in the same manner as that described in Example4 using BOC-D-proline (Peninsula Laboratories) at cycle 15 in lieu ofBOC-L-proline. Purification and characterization were performed asdescribed in Example 4.

EXAMPLE 6

Synthesis of Hirulog-10

Hirulog-10 has the formula:H--(D--Phe)--Pro--Arg--Sar-(Gly),--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH.The peptide was synthesized as in Example 4 using BOC-sarcosine (SigmaChemical Co., St. Louis, Mo.) at cycle 16. Purification andcharacterization were performed as described in Example 4.

EXAMPLE 7 Synthesis of Hirulog-11

Hirulog 11 has the formula: H--(D--Phe)--Pro--Arg--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--(3,5-diiodoTyr)--Leu--OH.This peptide is synthesized as in Example 4 usingBOC-3,5-diiodo-L-tyrosine (Sigma) at cycle 2. Purification andcharacterization is performed as described in Example 4.

EXAMPLE 8 Synthesis of Hirulog-12

Hirulog 12 has the formula: H--(D--Phe)--Pro--Arg--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr(OSO₃)--Leu--OH.This peptide is synthesized by reacting 1.0 mg of Hirulog-8 indimethylformamide (80 μl) with dicyclohexylcarbodiimide solution (1.25g/ml, 0.007 ml) and concentrated sulfuric acid (0.5 μl) at 0° C. for 10minutes. The reaction is stopped by addition of water (1.0 ml).

The reaction mixture may be subjected to reverse-phase HPLC employing anApplied Biosystems 150A Liquid Chromatographic System and an AquaporeRP-300 C₈ column (0.46×10 cm). The column is equilibrated in solvent A(0.1% TFA/water) and developed with an increasing concentration ofsolvent B (0.085% TFA/70% acetonitrile) from 0 to 50% over 45 minutes ata flow-rate of 1.0 ml/min. The effluent stream is monitored forabsorbance at 214 nm.

Purified Hirulog-12 is then neutralized to pH 7 by adding 0.1N NaOH. Itis then lyophilized and reconstituted in phosphate-buffered saline.

EXAMPLE 9

Inhibition of Thrombin-Catalyzed Hydrolysis of a p-NitroanilideSynthetic Substrate by Hirulog-8

We next analyzed the effects of Hirulog-8 on the humanα-thrombin-catalyzed hydrolysis of Spectrozyme TH(tosyl--Gly--Pro--Arg-p-nitroanilide; American Diagnostica, New York,N.Y.). Specifically, we measured the initial rate velocities in thepresence or absence of Hirulog-8 over a range of substrateconcentrations from 2.2 to 22 μM. The thrombin-catalyzed rate wasmonitored in a Cary 19 spectrophotometer at 405 nm and recordedcontinuously as a function of time. Kinetics were performed at roomtemperature (25±1° C.) in a 0.05M Tris, pH 7.5, 0.1M NaCl buffer.

For a typical enzyme reaction, 1.0 ml of buffer was added to both thesample and reference cuvettes. Thrombin (3.2×10⁻⁹ M, finalconcentration) and Hirulog-8 (0-4×10⁻⁸ M) were added to the samplecuvette prior to addition of Spectrozyme TH (2.2-22 μM). Immediatelyfollowing addition of substrate, the contents of the sample cuvette weremixed by use of a plastic pipette. The reaction was monitoredspectrophotometrically for 5-15 minutes.

Initial rate velocities at each substrate concentration were expressedas moles Spectrozyme TH hydrolyzed/sec/mole thrombin. This wasdetermined during the initial linear phase of the reaction (≦15% totalhydrolysis of substrate) by measuring the slope of the hydrolyticreaction. Lineweaver-Burke plots were constructed accordingly, byplotting the inverse of the initial velocity against the inverse of thesubstrate concentration. The results showed that humanα-thrombin-catalyzed hydrolysis of Spectrozyme TH had a V_(max) =17moles hydrolyzed/sec/mole thrombin and a K_(m) at 1.19×10⁻⁶ M. FIG. 3,panels A and B, demonstrates that increasing concentrations of Hirulog-8led to significant, dose-dependent increases in the K_(m), with slightincreases in the V_(max) for Spectrozyme TH hydrolysis. Therefore, theinhibition of the thrombin-catalyzed reaction by Hirulog-8 was carriedout by mixed competitive/non-competitive components with respect toSpectrozyme TH hydrolysis. The K_(i) of Hirulog-8 for α-thrombin wasdetermined using the equation: ##EQU1## is the slope of thethrombin-catalyzed reaction in the presence of Hirulog-8; [Hirulog-8] isthe molar concentration of peptide; ##EQU2## is the thethrombin-catalyzed reaction in the absence of inhibitor; and K_(i) isthe molar inhibitory constant for Hirulog-8 with human α-thrombin. TheK, for Hirulog-8 was calculated to be 1.95±0.11×10⁻⁹ M.

EXAMPLE 10 Specificity of Hirulog-8 for the Hirudin-Peptide Binding Siteand Active Site of Human α-Thrombin

Hirulog-8 was designed as an analogue that binds human α-thrombin viaits hirudin peptide binding 'site while blocking thrombin's catalyticsite. We tested the ability of Hirulog-8 to perform these functions byvarious studies described below.

The kinetics of Hirulog-8 inhibition of human γ-thrombin were studiedessentially as described above in Example 9 for human α-thrombin. Theγ-thrombin-catalyzed reaction toward Spectrozyme TH demonstrated aV_(max) =7.14 moles hydrolyzed/sec/mole thrombin and K_(m) = 1.1×10⁻⁶ M.These results confirm that γ-thrombin, a proteolytic form of thrombin,exhibits nearly complete catalytic competence, although this formessentially lacks clotting activity [S. D. Lewis et al., "CatalyticCompetence of Human α- and γ-Thrombins in the Activation of Fibrinogenand Factor XIII", Biochemistry, 26, pp. 7597-7603 (1987)]. Theinhibition of γ-thrombin by Hirulog-8 was examined over a range ofpeptide concentrations from 2.7×10⁻⁸ to 6.8×10⁻⁶ M. As shown below,Hirulog-8 exhibited an increased K_(i) of 3 orders of magnitude relativeto α-thrombin. This high K, toward 7-thrombin is due to the absence ofan intact anion binding exosite (ABE) in γ-thrombin [J. W. Fenton, II,et al , . "Anion-Binding Exosite of Human α-Thrombin and Fibrin(ogen)Recognition" Biochemistry, 27, pp 7106-12 (1988)]. γ-thrombin is formedby proteolysis of the B-chain of α-thrombin at Lys-149 and Arg-78.

The inhibition of human α-thrombin by Hirulog-8 was significantlyreduced in the presence of Sulfo-Tyr₆₃ -N-acetyl-hirudin₅₃₋₆₄ atconcentrations of 2.6 ×10⁻⁻⁶ M to 1.29×10⁻⁵ M. This is becauseSulfo--Tyr₆₃ -N-acetyl-hirudin₅₃₋₆₄ competes with Hirulog-8 for bindingto the ABE of thrombin.

This was also demonstrated by the addition ofphenylmethylsulfonyl-α-thrombin ("PMS-α-thrombin"; 18 nM, final) toreactions of Hirulog-8 with human α-thrombin. The addition of thismodified thrombin resulted in a substantial decrease in the ability ofHirulog-8 to inhibit α-thrombin. PMS-α-thrombin has an intact ABE, butis covalently derivatized at its active site. This modified thrombinsequesters the Hirulog-8 in the reaction mix and therefore reduces theamount of peptide available to inhibit intact, catalytically-activehuman α-thrombin.

We also performed studies of the effect of salt concentrations on theK_(i) of Hirulog-8 for thrombin as described above in Example 9. Wemeasured the K_(i) in the presence or absence of Hirulog-8 (11.5×10⁻⁹ M)in buffers containing 0.1, 0.25, and 0.5M NaCl. As shown in the tablebelow, inhibition of α-thrombin by Hirulog-8 increased at lower saltconcentrations. This result confirmed that the interaction of the highlyanionic hirudin peptide moiety of Hirulog-8 with the positively-chargedsite surrounding Lys-149 of thrombin is essential for Hirulog-8inhibition of thrombin-catalyzed hydrolysis of Spectrozyme TH.

    ______________________________________                                        Enzyme     Conditions     Hirulog-8, K.sub.i, nM                              ______________________________________                                        Human α-                                                                           0.05 M Tris, pH 7.5                                                                          1.95                                                thrombin   0.1 M NaCl (Buffer)                                                Human γ-                                                                           Buffer         1,080                                               thrombin                                                                      Human α-                                                                           Buffer + 2.6 μM                                                                           25.5                                                thrombin   Sulfo-Tyr.sub.63 - .sub.-- N-acetyl-                                          hirudin.sub.53-64                                                  Human α-                                                                           Buffer + 12.9 μM                                                                          >2,000                                              thrombin   Sulfo-Tyr.sub.63 - .sub.-- N-acetyl-                                          hirudin.sub.53-64                                                  Human α-                                                                           Buffer + PMS-  9.90                                                thrombin   α-thrombin                                                   Human α-                                                                           0.05 M Tris, pH 7.5                                                                          2.09                                                thrombin   0.25 M NaCl                                                        Human α-                                                                           0.05 M Tris, pH 7.5,                                                                         3.72                                                thrombin   0.5 M NaCl.                                                        ______________________________________                                    

EXAMPLE 11 Anticoagulant Activity of Hirulog-8: Comparison to Hirudinand Sulfo-Tyr₆₃ -N-Acetyl-hirudin₅₃₋₆₄

We studied the anticoagulant activity of Hirulog-8 using pooled, normalhuman plasma (George King Biomedical, Overland Park, Kans.) and aCoag-A-Mate XC instrument (General Diagnostics, Organon Technica,Oklahoma City, Okla.). Activity was monitored using the activatedpartial thromboplastin time (APTT) assay with CaCl₂ and phospholipidsolutions obtained from the manufacturer. Hirulog-8, hirudin, orSulfo-Tyr₆₃ -N-acetyl-hirudin₅₃₋₆₄ was then added to the APTTdetermination wells at a final concentrations of 10 to 32,300 ng/ml in atotal volume of 25 μl prior to addition of 100 μl of plasma.

The control APTT (absence of inhibitor) was 29.6 sec (mean, n=8,SEM<0.5%). FIG. 4 shows the results of these dose-dependency studies.Hirulog-8 was 2 to 3 times more potent than hirudin and 100 to 150 timesmore potent than Sulfo-Tyr₆₃ -N-acetylhirudin₅₃₋₆₄, Both Hirulog-8 andhirudin increased the APTT of plasma to values which were too high to bemeasured. This is in contrast to Sulfo-Tyr₆₃ -N-acetylhirudin₅₃₋₆₄,which exhibited a saturable dose-response in the APTT to 200-250% ofcontrol valves [J. M. Maraganore et al., J. Biol. Chem., 264, pp.8692-98, (1989)]. This result showed that Hirulog-8 can block the activesite of thrombin in plasma, as well as in vitro in chromogenic assays,in a manner similar to hirudin.

EXAMPLE 12 Inhibition of Thrombin Induced Platelet Activation ByHirulog-8

Thrombin-induced platelet activation studies are performed at 37° C.using a Biodata PAP. Platelet Aggregometer. Platelet-rich plasma (PRP)is obtained from normal, healthy, volunteers who have not taken anymedication altering platelet function for at least one week prior tostudy. PRP is prepared as described by J. A. Jakubowski et al.,"Modification of Human Platelet by a Diet Enriched in Saturated orPolyunsaturated Fat", Atherosclerosis, 31, pp. 335-44 (1978). Varyingconcentrations of Hirulog-8 (0-500 ng/ml in 50 μl water) are added to0.4 ml of pre-warmed (37° C.) PRP. One minute later, we add humanα-thrombin to the platelet suspension to a final concentration of 0.2,0.25 or 0.5 units/ml total assay volume. Aggregation is monitored as anincrease in light transmission for 5 minutes following the addition ofthrombin. We then calculate %Inhibition as (%aggregation_(sample))/(%aggregation_(control))×100. This study shows that Hirulog-8 blocksthrombin-induced platelet activation in vitro.

EXAMPLE 13 Use of Hirulog- 8 in Thrombus Imaging

Hirulog-8 is modified by covalent attachment of an ¹²³ I-containingchemical group. Specifically, Hirulog-8 (as prepared in Example 4) isreacted with ¹²³ I-Bolton Hunter Reagent (New England Nuclear, Boston,Mass.) in 0.1M sodium borate, pH 9.0. The ¹²³ I-labelled molecule (witha specific activity of >5 μCi/μg) is then desalted on a Biogel P2 columnwhich is equilibrated in a phosphate-buffered saline.

Ex vivo imaging of experimental thrombi is performed essentially asdescribed by T. M. Palabrica et al., "Thrombus Imaging in a PrimateModel with Antibodies Specific for an External Membrane Protein ofActivated Platelets", Proc. Natl. Acad. Sci. USA, 86, pp. 1036-40(1989). Specifically, imaging is performed in baboons using an externalTicoflex shunt between the femoral artery and femoral vein. Anexperimental thrombus is formed by placement of a segment of preclottedDacron graft in the shunt. ¹²³ I-labelled thrombin inhibitor is injectedin the venous portion of the Ticoflex shunt. Serial anterior images arethen obtained for 0.5 to 1 hour using an Ohio Nuclear Series 100 GammaCamera with a PDP-11/34 computer. The kinetics of ¹²³ I-thrombininhibitor uptake by the graft and the blood pool are derived from theradionuclide images thus obtained.

The same technique may be used to obtain ex vivo images of a deep venousthrombus caused by stasis in the femoral vein of baboons. Because ¹²³I-Hirulog-8 binds to thrombin with high specificity, the use of thismolecule allows precise ex vivo images of thrombi. Also, the small sizeof Hirulog-8, in contrast to native hirudin or antibodies to thrombin,provides the potential that the radiolabelled thrombin inhibitor willyield images of platelet-bound thrombin and meizothrombin, as well asthrombin contained in the fibrin clot.

EXAMPLE 14 Anti-Metastatic Activity of Thrombin Inhibitors

The anti-metastatic activity of the thrombin inhibitors of thisinvention, preferably Hirulog-8, is assayed using sarcoma T241 cells [L.A. Liotta et al., Nature, 284, pp. 67-68 (1980)] and syngeneic C57BL/6mice (Jackson Laboratory, Bar Harbor, ME). The mice are injected eitherintravenously or subcutaneously with 0-250 g/kg of Hirulog-8, preparedas in Example 4, followed by injection with 10⁴ -10⁶ T241 tumor cells.After 15 days, the animal is sacrificed and lung tumor colonies arequantitated. Anti-metastatic activity of Hirulog-8 is measured aspercent reduction in tumor colonies compared to placebo-treated controlmice. Hirulog-8 demonstrates anti-metastatic activity in this assay.

EXAMPLE 15 Inhibition of Endothelial Cells by a Thrombin Inhibitor

The ability of the thrombin inhibitors of this invention to preventthrombin-induced synthesis of platelet activating factor (PAF) isassayed using cultured human umbilical vein endothelial cells (HUVECs).HUVECS are extracted from human umbilical cords by collagenase digestionaccording to established procedures [M. A. Gimborne, Jr., "Culture ofVascular Endothelium", Prog. Hemost, Thromb., 3, pp. 1-28 (1976)].HUVECs are grown to confluence in a 96-well microtiter plate in thepresence of [³ H]-acetate. Cells cultured in this manner produce [³H]-acetyl-PAF, which may be quantitated by extraction of HUVEC membranephospholipids.

Hirulog-8 (0-1 μg/ml) is added to the [³ H]-acetate loaded HUVECs 1minute prior to the addition of thrombin (final concentration of IU/ml). Cells are incubated for 5 minutes and the supernatant is thenremoved. Medium containing 0.1% gelatin, 50 mM acetic acid in methanol(2:1 v/v) is then added to the HUVECs. PAF is then extracted andquantified using conventional techniques [T. M. Mcintyre et al.,"Cultured Endothelial Cells Synthesize Both Platelet-Activating Factorand Prostacyclin in Response to Histamine, Bradykinin and AdenosineTriphosphate", J. Clin. Invest., 76, pp. 271-80 (1985)]. The IC₅₀ valuesare then calculated. Hirulog-8 inhibits the synthesis of PAF by HUVECsin this assay.

The effect of Hirulog-8 on thrombin-induced polymorphonuclear leukocyte(PMN) adhesion to HUVECs may be demonstrated as follows. HUVECs aregrown to confluence in MEM containing 1% fetal calf serum in 24wellcluster plates. The medium is then removed, the cells are washed twotimes with fresh, serum-free medium and incubated in the same medium for10-30 minutes at 37° C. to remove serum products. PMNs (2.5×10⁶ in 1ml), which are pre-equilibrated at 37° C., are then added to each well.The PMNs are allowed to settle onto the HUVEC monolayer for 2 minutes.Hirulog-8 (5 μg/ml) or saline is added to each well, immediatelyfollowed by the addition of α-thrombin (0.1 or 1 U/ml). The cells areincubated for 5 minutes at 37° C., washed twice and then examined byphase-contrast microscopy. Adherent PMNs are counted directly. Samplesincubated with Hirulog-8 have significantly fewer adherent PMNs thanthose treated with saline.

EXAMPLE 16

Synthesis of Hirulog-13

Hirulog-13 has the formula: H--(D--Phe)--Pro--Arg--Pro--(Gly)₂--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. Wesynthesized, purified and characterized this peptide essentially asdescribed in Example 4, except that only one cycle of BOC-glycylglycinewas employed to produce the diglycine segment.

EXAMPLE 17 Synthesis of Hirulog-14

Hirulog-14 has the formula: H--(D--Phe)--Pro--Arg--Pro--(Gly)₅--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu- Tyr--Leu--OH.Hirulog-14 was synthesized, purified and characterized using methodsdescribed in Example 4, except that one cycle of BOC-glycine additionwas employed following the two cycles of BOC-glycylglycine addition toproduce the pentaglycine segment.

EXAMPLE 18 Synthesis of Hirulog-15

Hirulog-15 has the formula: H--(D--Phe)--Pro--Arg--Pro--(Gly)₆--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH.Hirulog-15 was synthesized, purified and characterized using methodsdescribed in Example 4, except that three cycles of BOC-glycylglycineaddition were employed to prepare the hexaglycine segment.

EXAMPLE 19 Synthesis of Hirulog-16

Hirulog-16 has the formula: H--(D--Phe)--Pro--Arg--Pro--(Gly)₇--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH.Hirulog-16 was prepared, purified and characterized as described inExample 4, except that four cycles of BOC-glycylglycine addition wereused to prepare the octaglycine segment.

EXAMPLE 20 Synthesis of Hirulog-7

Hirulog-17 has the formula:H--(D--Phe)--Pro--Arg--Pro--Gly--Gly--Glu--Gly-His--Gly--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu-Tyr--Leu--OH.Hirulog-17 was synthesized essentially as described in Example 4, exceptthat a Gly--Gly--Glu--Gly-His--Gly replaced the Gly₄ segment present inHirulog-8. This Sequence was added on to the growing peptide chain bythe consecutive additions of BOC-glycine, BOC-L-histidine, BOC-glycine,BOC-L-glutamic acid and BOC-glycylglycine at cycles 13-17 of synthesis.Purification and characterization were performed as described in Example4.

EXAMPLE 21 Synthesis of Hirulog-18a, -18b and -18c

Hirulog-18a has the formula:H--(D--Phe)--Pro--(β-homoarginine)-(Gly)s--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu-Tyr--Leu--OH.Hirulog-18b has the formula:H--(D--Phe)--Pro-(β-homoarginine)--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu-Tyr--Leu--OH.Hirulog-18c has the formula:H--(D--Phe)--Pro--(β-homoarginine)--Val(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. Wesynthesized Hirulog-18a using a mixed homogeneous/solid-phase procedure.Residues 5-20 were prepared by solid-phase synthesis, as described inExamples 4 and 17. The resulting resin-linked intermediate was reactedwith a BOC-β-homoarginine--Gly protected intermediate, which wassynthesized in the multi-step reaction scheme depicted below anddescribed immediately thereafter. ##STR4##

N.sup.α -BOC-N^(g) -Tos-ArginineDiazomethylketone

We stirred 10 g (13.4 mmoles) of N.sup.α -BOC-N⁹ -Tos-arginine (Bachem,Torrance, Calif.) and 2.1 ml (19.1 mmole) of N-methylmorpholine(Aldrich, Milwaukee, Wis.) in 100 ml anhydrous tetrahydrofuran (THF)under argon for 5 minutes at room temperature. The solution was thencooled to -15° C. and 2.8 ml (21.6 mmol) of isobutylchloroformate(Aldrich) was added. We continued to stir the reaction mixture at -15°C. for 5 minutes, and then filtered it through a pad of Celite/MgSO₄. Wenext added the filtrate to an ice-cold ethereal solution of diazomethane(150 mM, generated from 32.4 g Diazald; Aldrich). The solution wasstirred and allowed to gradually reach ambient temperature overnight.The solvent was then removed in vacuo and the residue dissolved in 200ml chloroform. We then washed the organic solution successively with 200ml of saturated NaHCO₃, followed by 200 ml of saturated NaCl, dried itover anhydrous MgSO₄, and concentrated it again to an oily residue. Theresidue was then purified by flash chromatography on a 4×17 cm column ofsilica gel using a step gradient of acetone in chloroform (10% acetonein 2 1 chloroform, followed by 20% acetone in 3 1 chloroform). Fractionsof 25 ml were collected. Aliquots of each fraction were assayed bythin-layer chromatography (TLC). Fractions containing the desiredproduct were pooled and evaporated to dryness. The product,diazomethylketone, was purified as a pale yellow foam (6.54 g).

N.sup.α -BOC-N^(g) -Tos-β-Homoarginine Methylester

We dissolved the diazomethylketone prepared above in 100 ml of anhydrousmethanol and refluxed that solution under argon while a solution ofsilver benzoate catalyst (165 mg in 400 μl triethylamine) was addeddropwise. After 30 minutes, the refluxing solution was cooled to roomtemperature, slurried with Norit, and filtered through Celite. Thesolvent was then removed in vacuo and the oily residue purified by flashchromatography over silica gel. Elution was achieved with 4 l of 10%acetone in chloroform. The desired product, β-homoarginine methylester,was thus purified as a light tan foam (6.43 g).

N.sup.α -BOC-N^(g) -Tos-β-Homoarginine

We dissolved all of the above methyl ester in 100 ml of methanol andthen reacted it with a solution of LiOH (1.48 g in 50 ml water)overnight at room temperature under argon with constant stirring. Weremoved the methanol in vacuo, dissolved the residue in water and washedit with ethyl acetate. We next added saturated citric acid until thesolution reached a pH of 4. We then extracted the resulting carboxylicacid into ethyl acetate. The extraction was repeated at pH 3, and thecombined organic phases were dried over MgSO₄ and concentrated in vacuo.The resulting crude acid was recovered as a white foam (4.9 g). The acidwas further purified on a Vydac C₁₈ reverse-phase HPLC column, asdescribed in Example 4, except that the effluent stream was monitored at214 nm. Following lyophilization of the desired fractions, the product,N.sup.α -BOC-N^(g) -Tos-β-homoarginine, was recovered as a whiteamorphous solid.

A sample of the N.sup.α -BOC-N^(g) -Tos-β-homoarginine was hydrolysed inHF and used as a standard for amino acid analysis. The retention time ofβ-homoarginine was identical to that of arginine, but the intensity ofthe peak was considerably lower, as expected.

N.sup.α -BOC-N^(g) -Tos-β-Homoargininylglycine Benzylester

We next combined 4.06 g (9.2 mmoles) of the above carboxylic acid with2.04 ml of N-methylmorpholine in 25 ml of anhydrous THF. The mixture wasstirred under argon at -5° C. A chilled solution ofisobutylchloroformate (2.4 ml in 25 ml THF) was then added dropwise tothe solution over 10 minutes. Following this addition, the reactionmixture was stirred for 12 minutes at -5° C. For Hirulog-18a we thenadded a solution of glycine benzyl ester (4.9 g in 40 ml THF; 27.6mmoles), and allowed the reaction mixture to come to room temperature.The solvent was then removed in vacuo and the resulting residuedissolved in 100 ml ethylacetate. The solution was extractedsuccessively with 100 ml each of saturated NaHCO₃ and saturated NaCl,dried over MgSO₄, and concentrated in vacuo. The resulting crudedipeptide ester was purified on a 4×20 cm silica gel column with amethanol step gradient in chloroform containing 10 drops NH₄ OH per 100ml (2 1 of 1% methanol in chloroform, followed by 3 of 2% methanol inchloroform). Fractions (25 ml) were collected, assayed by TLC and thosecontaining product were pooled and the solvent removed in vacuo. Theresulting product, N.sup.α -BOC-N^(g) -Tos-β-homoargininylglycinebenzylester, was isolated a white foam (3.9 g).

For Hirulog-18b and -18c, the above reaction was identical except forthe following modifications: For Hirulog-18b, the glycine benzyl esterwas replaced by proline benzyl ester and the reaction was run on a 1.8mmole scale. For Hirulog-18c, the glycine benzyl ester was replaced withvaline benzyl ester and the reaction was run on a 3.0 mmole scale.

N.sup.α -BOC-N^(g) -Tos-β-Homoargininylglycine

The above benzyl ester was dissolved in 50 ml methanol and hydrogenatedat atmospheric pressure over 1.0 g of 10% palladium/carbon for 17 h. Theresulting solution was filtered through Celite and the solvent removedin vacuo. The reaction yielded 2.9 g of crude N.sup.α -BOC-N^(g)-Tos-β-homoargininylglycine, which was purified on a Vydac C₁₈ HPLCcolumn as described above.

The above N.sup.α -BOC-N^(g) -Tos-β-homoargininylglycine (1.02 g) wasdissolved in 1 ml anhydrous DMF and cooled in an ice bath. We then addedto this solution successively, 5.5 ml of 0.5M hydroxybenztriazole in DMF(Applied Biosystems Inc, Foster City, Calif.) and 5.5 ml of 0.5Mdicyclohexylcarbodiimide in CH,Cl, (Applied Biosystems). After 1 hour,the cold suspension of symmetrical anhydride of the dipeptide unit wasthen rapidly filtered through a plug of glass wool to remove thedicyclohexyl urea.

Meanwhile, a suspension of N-BOC-(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--O--PAM(0.2 mmol in CH₂ Cl₂) was activated by standard peptide synthesismethods. A Kaiser test on the resulting product indicated a freeterminal amino group.

The activated β-homoarginylglycine dipeptide was then coupled to theresin-bound hexadecapeptide. The resulting octadecapeptide was thencoupled, successively, with N-BOC-Pro and N-BOC-(D--Phe) using standardcoupling procedure. The resulting peptide, Hirulog-18a, was purified andcharacterized as described in Example 4.

A similar protocol was carried out for the synthesis of Hirulog-18b andHirulog-18c.

EXAMPLE 22 Synthesis of Hirulog-19

Hirulog-19 has the formula: H--(D--Phe)--Pro--Arg--[psiCH₂ NH]--(Gly)₅--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH.Residues 4-20 of this peptide were assembled by solid-phase peptidesynthetic procedures as described in Example 4. The next residue added,N.sup.α --BOC--N⁹ -tosyl-argininal, was prepared as depicted anddescribed below. ##STR5##

N.sup.α -BOC-N^(g) -Tos-Arginal

N.sup.α -BOC-N^(g) -Tos-arginine (Bachem Inc.; 10 g) was added to 80 mlof anhydrous THF and the suspension cooled to 0°-5° C. We then added 1,1'-carbonyldiimidazole (Aldrich; 3.61 g) all at once and continuedstirring for 20 minutes. The resulting clear solution was partiallyimmersed in a dry ice/acetone bath to maintain a temperature of -20° to-30° C. during the dropwise addition of a suspension of lithium aluminumhydride (Aldrich; 1.8 g in 80 ml THF) over 45 minutes with constantstirring. The reaction was stirred an additional 30 minutes at -20° C.and was then quenched by the dropwise addition of 63 ml of 2N HCl at-10° C. We filtered the resulting solution through a medium scinterglass funnel and concentrated the resulting filtrate in vacuo.

The resulting crude aldehyde, recovered as a white foam (11.5 g), wassuspended in 100 ml of chloroform, washed with water (2×50 ml) and theorganic layer then dried over sodium sulfate and concentrated in vacuo.The crude aldehyde (7.7 g) was dissolved in 100 ml chloroform andpurified by flash chromatography over a 5×20 cm flash column containing350 ml silica gel (Merck Grade 60, 230-400 mesh, 60 Å). Elution wasachieved using a step gradient of. 0.5% methanol in 500 ml chloroform,1% methanol in 1 l chloroform, and 1.5% methanol in I 1 chloroform. Thisprocedure yielded 8.9 g of N.sup.α -BOC-N^(g) -Tos-argininal.

The N.sup.α -BOC-N^(g) -Tos-argininal (258 mg) was then added to theresin-bound (Gly)₅--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--O--PAMunder solid-phase reductive alkylation conditions (40 mg sodiumcyanoborohydride for 24 hours) using the method of D. H. Coy et al.,"Solid-Phase Synthesis of Peptides" In Peptides, Vol. 8, pp. 119-121(1978). Following reaction of the resin-linked peptide with theprotected argininal, the peptide synthesis was completed with a cycle ofBOC-proline incorporation and a cycle of BOC-(D-phenylalanine)incorporation. After completion of the synthesis, Hirulog-19 wasdeprotected and uncoupled from the resin as described in Example 4.

Hirulog-19 was purified by reverse phase HPLC employing an AppliedBiosystems 151A liquid chromatographic system and an Aquapore C₈ column(10×22 cm). The column was equilibrated in 1 part 70% acetonitrile/30%water containing 0.85% TFA (Buffer B) and 4 parts water containing 1%TFA (Buffer A). The column was developed with a linear gradient ofincreasing Buffer B concentration (20-50%) over 120 minutes at a flowrate of 4.0 ml/minute. The effluent stream was monitored for absorbanceat 214 nm and fractions were collected manually. Further purificationwas carried out under isocratic conditions using 20% Buffer B/80% BufferA.

EXAMPLE 23 Synthesis of Hirulog-21

Hirulog-21 has the formula: H--(D--Phe)--Pro--Arg--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--(Gly)₂--Lys--OH. Hirulog-21 was synthesized using methods described in Example4, using the appropriate BOC-amino acids. Purification andcharacterization of Hirulog-21 were achieved by the methods described inExample 4.

EXAMPLE 24 Synthesis of Hirulog-25

Hirulog-25 has the formulaH--(D--Phe)--Pro--(4-Argininyl-2,2-difluoro)malonylglycyl-(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. Thehexadecapeptide, (Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu, wassynthesized as previously described and left bound to the resin. Thenext residue, (3--Argininyl-2,2-difluoro)malonylglycine is synthesizedin the reaction scheme depicted and detailed below. ##STR6##

1-[(2'-carboethoxy-1',1'-difluoro)ethyl]N.sup.α -BOC-N^(orn)-benzyl-N^(orn) -CbzOrnithinol

A solution of 3.1 g (7.1 mmoles) N.sup.α -BOC-N^(orn) -Benzyl-N^(orn)-CbzOrnithinal [F. Salituro et el., "Inhibition of Aspattic ProteinasesBy Peptides Containing Lysine and Onithine Side Chain Analogues ofStatine", J. Med. Chem,, 30, pp. 286-95 (1987)], and 1.56 ml (9.23mmoles) ethylbromodifluoroacetate in anhydrous 15 ml THF was added over90 minutes to a refluxing suspension of 786 mg Zn powder (Fluke) in 15ml THF under argon. After 4 hours of reflux and 2 hours at roomtemperature, the mixture was cooled and 15 partitioned between 200 mleach of ethyl acetate and saturated NaCl/KHSO₄. The organic phase wasisolated, dried over MgSO₄ and concentrated in vacuo. The resulting oilyresidue was purified on silica gel, using CHCl₃ :methanol (90:10) plus100 drops/l NH₄ OH as eluant.

1-[(2'-Carboethoxy-1',1'-difluoro)ethyl]N.sup.α -BOC-OrnithinoltertButyldimethylsilyl Ether

The resulting compound, 1-[(2'-carboethoxy-1'-1'-difluoro)ethyl]N.sup.α-BOC-N^(orn) -benzyl-N^(orn) -CbzOrnithinol, is then reacted with 5equivalents of tert-butyldimethylsilyl chloride and 10 equivalents ofimidazole in anhydrous DMF at 35° C., following the procedure of E. J.Corey et al., "Protection of Hydroxyl Groups as tertButyldimethylsilylDerivatives", J. Amer. Chem. Soc., 94, pp. 6190-91, (1972). Theorthogonally protected amine is then dissolved in methanol andhydrogenated over Pd(OH)₂ at 30 psi for 18 hours. The catalyst is thenremoved by filtration and the filtrate concentrated in vacuo to produce1-[(2'-carboethoxy-1'-1'-difluoro)ethyl]N.sup.α-BOC-Ornithinoltert-butyldimethylsilyl ether.

1-[(2 '-Carboethoxy-1',1'-difluoro)ethyl]N.sup.α -BOC-N^(g)-Tos-Arqininol-tertButyldimethylsilyl Ether

The above-prepared compound is then reacted with 6.8 equivalents each of1-guanyl-3,5-dimethylpyrazole and triethylamine in water at 105° C. for24 hours. The mixture is then lyophilized and the residue subjected topreparative HPLC as described in Example 4. Fractions containing thedesired guanidinium compound (assayed by TLC) are pooled and dried invacuo. The residue is dissolved in H₂ O: acetone (1:4), cooled in an icebath and adjusted to pH 12 with 50% w/v NaOH. To this solution we add asolution of 3 equivalents of paratoluene sulfonylchloride in acetoneover 60 minutes, while maintaining the pH at 11-12 with NaOH. Thesolution is allowed to warm to room temperature and is stirredovernight. The acetone is then removed in vacuo and the remainingaqueous solution is washed with ether. The ether layer is removed andback extracted with saturated NaHCO₃. The aqueous phases are combinedand acidified to pH 3 with 2N HCl. The resulting acid solution is thenextracted two times with ethyl acetate, dried and concentrated in vacuoto yield the desired product.

1-[(2'-Carboxy-1', 1'-difluoro)ethyl]N.sup.α -BOC-N^(g) -Tos-Argininol

The resulting compound, 1-[(2'-carboethoxy-1'-1'-difluoro)ethyl]N.sup.α-BOC-N^(g) -Tos-Argininoltertbutyldimethylsilyl ether, is desilylated bytreatment with 3 equivalents of tetra-β-butylammonium fluoride in THF atroom temperature, as described in E. J. Corey et al., supra. Thecompound produced by this process is then saponified by treatment with2.5 equivalents of LiOH in methanol/water at room temperature overnightunder argon. The reaction mixture is then washed with ethyl acetate andacidified with citric acid to pH 4. We extract the resulting acid intoethyl acetate, dry the organic phase and concentrate it in vacuo. Thecrude acid is then purified on a Vydac C₁₈ reverse-phase HPLC columnunder the conditions described in Example 4.

1-[(2'-Carboxy-1',1'-difluoro)ethyl]N.sup.α -BOC-N^(g) -Tos-Argininone

The alcohol function of the above compound is converted to the ketone bythe addition of one equivalent of pyridinium dichromate in CH₂ Cl₂containing 0.5% glacial acetic acid in the presence of molecular sieves[N. Peet et al., "Synthesis of Peptidyl and Fluoromethyl Ketones andPeptidyl α-Keto Esters as Inhibitors of Porcine Pancreatic Elastase,Human Neutrophil Elastase, and Rat and Human Neutrophil Cathepsin G", J.Med. Chem,, 33, pp. 394-407 (1990)]. After stirring under argon for 15hours, the reaction mixture is filtered and the solvent removed invacuo. The resulting 1-[(2'-carboxy-1',1'-difluoro)ethyl]N.sup.α-BOC-N^(g) -Tos-Argininone is recovered as an oily residue and thenpurified on HPLC according to the conditions specified in Example 4.

The free carboxylic acid is converted to the symmetrical anhydride andreacted with resin-bound hexadecapeptide as described in Example 21. Thetwo N-terminal residues of Hirulog-25, BOC--Pro and BOC--(D--Phe), areadded under standard peptide synthesis conditions and the resultingpeptide is then cleaved with HF.

EXAMPLE 25 Synthesis of Hirulog-26

Hirulog-26 has the formula:H--(D--Phe)--Pro--Argoxopropionylglycyl--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. Thehexadecapeptide, (Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu, wassynthesized as previously described and left bound to the resin. Thenext residue, N.sup.α -BOC-argoxopropionylglycine, is synthesized by thereaction scheme depicted and described below. ##STR7##

3-(CbzAmino)-2-oxo-3-{3-[(N^(g)-Tos)guanidinyl]propyl}ditertbutylMalonate

We prepared a batch of N.sup.α -Cbz-N^(g) -Tos-ArginineDiazomethylketone in the same manner as the preparation of N.sup.α -BOC-N^(g)-Tos-ArginineDiazomethyl ketone described in Example 21, except for thesubstitution of N.sup.α -Cbz-N^(g) -Tos-Arginine for N.sup.α -BOC-N^(g)-Tos-Arginine. We dissolved 4.5 mg of N.sup.α -Cbz-N^(g)-Tos-ArginineDiazomethyl ketone in 200 ml of CH₂ Cl₂ in a flask andcooled the solution to -70° C. in a dry ice/acetone bath with stirring.Anhydrous HBr gas was then bubbled through the solution at a moderateflow rate for 15 minutes. The solution was stirred for an additional 15minutes at -70° C. and then concentrated in vacuo. The resultingproduct, N.sup.α -Cbz-N^(g) -Tos--Arg--COCH₂ Br, was recovered as 5.0 gof yellow crystals.

Meanwhile, a suspension of sodium hydride (36 mg; 80% dispersion in oil)in 1 ml DMF and 1.2 ml hexamethylphosphoramide ("HMPA") was added to asolution of 259 mg ditertbutoxymalonate in 4 ml DMF. The mixture wasstirred at room temperature for 40 minutes and was then added dropwise,over 20 minutes, to a solution of 1 mmole N.sup.α -Cbz-N^(g)-Tos--Arg-COCH₂ Br, in 1 ml DMF/0.13 ml HMPA. The reaction was allowedto proceed for 3 hours, after which time the solution was poured into 50ml water and extracted with 2×50 ml ethyl acetate. The organic phase wasisolated, dried and concentrated in vacuo to an oily residue. Theresidue was subsequently purified on a 3×10 cm silica gel column whichwas eluted successively with 400 ml of 5% acetone in chloroform, 400 mlof 10% acetone in chloroform and 200 ml of 20% acetone in chloroform.Fractions (25 ml) were collected and assayed by TLC. Fractionscontaining the desired product were pooled and concentrated to produce3-(CbzAmino)-2-oxo-3-{3-[(N^(g) -Tos)guanidinyl] propyl}-di-tertButylmalonate.

5- (N.sup.α -CbzAmino)-4-oxo-5-{3-[(N^(g)-Tos)quanidinyl]propyl}pentanoylglycine Benzyl Ester

The above di-t butyl ester is stirred in 1.2 equivalents of 1N HCl for 2hours at room temperature. It is then decarboxylated in excess pyridineat 100° C. for 15 minutes. The solvent is then removed in vacuo, and theresidue purified by silica gel chromatography, as described above. Theresulting carboxylic acid is acylated with glycine benzyl esteraccording to the method described in Example 21.

5-(Amino]-4-oxo-5-{3-[(N^(g) -Tos) guanidinyl]propyl}pentanoylglycine

The resulting ester is dissolved in 500 ml methanol and hydrogenatedovernight at 1 atmosphere of hydrogen gas over 600 mg of 10%palladium-carbon catalyst. The reaction mixture is then filtered throughCelite and concentrated in vacuo to a solid residue (155 mg). Theresulting amino acid is then purified by HPLC, using the conditionsdescribed in Example 4.

5-(N.sup.α -BOCAmino)-4-oxo-5-{3-[(N^(g) -Tos)guanidinyl]propyl}pentanoylglycine

The above amino acid is converted to its corresponding BOC derivative bydissolving in dioxane/water (2:1, v/v) and cooling to 0° C. withstirring. The pH is adjusted to 10 with 0.1N NaOH and. then 1.1equivalents of di-tert-butyl dicarbonate (in dioxane) are added. Thereaction is stirred at 0° C. to 20° C. for 4 hours and then isevaporated in vacuo. The residue is then partitioned between ethylacetate/1% citric acid (2:1). The organic phase is isolated, extractedonce with 1% citric acid, and then 3 times with saturated NaCl. Theorganic phase is dried over MgSO₄, filtered and concentrated in vacuo toobtain the BOC-protected product.

The resulting protected psuedopeptide free carboxylate is then coupledto the resin-bound hexadecapeptide using standard peptide synthesistechniques. This is followed by the sequential addition of BOC--D--Pheand BOC--Pro to the resin-bound peptide. The completed Hirulog-26 isthen cleaved from the resin, deprotected and purified as described inExample 4.

EXAMPLE 26 Synthesis of Hirulog-27

Hirulog-27 has the formula H--(D--Phe)--Pro--Arg--(CO--CH₂)--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. The(Gly)₄ --Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leuhexadecapeptide was synthesized as previously described and left boundto the resin. The remaining portion of the molecule was synthesized bythe reaction scheme depicted and described below. ##STR8##

N.sup.α --Cbz--N⁹ --Tos-Arginine(COCH₂)proline Benzyl Ester

We dissolved 720 mg of proline benzyl ester (HCl salt) in 25 ml THF.This solution was then cooled to -78° C. in an acetone/dry ice bath withstirring under argon. We then added lithium diisopropylamide (8.0 ml ofa 0.75M hexane suspension) and stirred for an additional 5 minutes. Tothis we added 1.08 g N.sup.α -Cbz-N^(g) -Tos-ArginineBromomethyl Ketonein 10 ml THF, prepared as described in Example 25, dropwise over 20minutes. The reaction was stirred for an additional 5 minutes and thesolution was then allowed to warm to room temperature with stirring. Wequenched the reaction by adding 10 ml of saturated NaCl, allowed thephases to separate and isolated the organic phase. This phase was thendried over MgSO₄, filtered and evaporated in vacuo.

N.sup.α -BOC-N^(g) -Tos-Arginine(COCH₂)proline

The above benzyl ester (1.3 g) was hydrogenated using thepalladium-carbon procedure described in Example 25. The resultingpseudodipeptide was BOC-protected by the procedure described in Example25 to produce the desired product.

The purified, protected pseudodipeptide was then coupled with theresin-linked hexadecapeptide by standard peptide synthesis techniques.Hirulog-27 was deprotected, cleaved from the resin and purified by thetechniques described in Example 4.

EXAMPLE 27 Synthesis of Hirulog-28

Hirulog-28 has the formula: H--(D--Phe)--Pro--Arg(CH₂ N)--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. The(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--O--PAMhexadecapeptide was synthesized as previously described and left boundto the resin. The remaining portion of the molecule was synthesized bythe reaction scheme depicted and described below. ##STR9##

N.sup.α -BOC-N^(g) -Tos-Arginine[psiCH₂ N]Proline Benzyl Ester

One gram of crushed 3Å molecular sieves (Aldrich) was added to a stirredsolution of 5.25 g proline benzyl ester free base (Schweizerhall, Inc.)in 10 ml anhydrous THF and 2 ml anhydrous ethanol under argon at roomtemperature. We added 1.45 ml of 5N methanolic HCl and 1.5 g of N.sup.α-BOC-N^(g) -Tos-Argininal (prepared as described in Example 22) to thismixture and stirred for i hour. An 85 mg portion of sodiumcyanoborohydride was added to the mixture and then, an hour later, asecond 85 mg portion of sodium cyanoborohydride was added. The reactionwas then stirred for 20 hours and filtered. We added 1 ml water and 0.9ml 1N HCl to the liltrate with stirring and then concentrated thesolution in vacuo to yield 6.2 g of-N.sup.α -BOC-N^(g) --Arg[psiCH₂N]--Pro-benzyl ester, as a clear oil.

The oil is further purified by flash chromatography over a 5 cm flashcolumn containing 350 ml silica gel (Merck Grade 60, 230-400 mesh, 60Å). The product was obtained by succesive elution with 0.25%, 0.75% and1.5% methanol in chloroform.

N.sup.α -BOC-N^(g) -Tos-Arginine[psiCH₂ N]Proline

The resulting benzyl ester is hydrogenated over palladium-carbon andpurified, as described in Example 25. This process yielded 160 mg ofN.sup.α -BOC-N^(g) -Arg[psiCH₂ N]--Proline free acid, which was furtherpurified using the HPLC chromatography system described in Example 4,except elution was achieved with an isocratic 26% Buffer B/74% Buffer Asystem, previously described in Example 22. The final yield of dipeptidewas 86 mg.

The dipeptide is then coupled to the resin-bound hexadecapeptide,followed by a cycle of BOC--Pro incorporation and a cycle ofBOC-(D--Phe) incorporation. Deprotection, cleavage and purification ofthe fully synthesized Hirulog-28 is achieved by the method described inExample 4.

EXAMPLE 28 Synthesis of Hirulog-29

Hirulog-29 has the formula: 4-chloroisocoumarino-3-carboxyethoxy-(Gly)₅--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. The(Gly)₅ --Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leuheptadecapeptide was synthesized as previously described and left boundto the resin. The 4-chloroisocoumarino-3-carboxyalkoxy moiety wassynthesized by the reaction scheme and methods described below.##STR10##

Ethyl 2-bromo-Homopthalate

We mixed homopthalic acid (10.0 g), 2-bromoethanol (21.0 g) and benzene(200 ml). We then added 12-15 drops of sulfuric acid and heated toreflux for 2.5 hours. The solution was then filtered and concentrated invacuo. The residue was washed with 250 ml ether/hexane (1:1) and wasfiltered onto a scintered glass funnel. The resulting light brown solidwas vacuum dried to obtain approximately 15.0 g of product.

4-chloro-3-[2-bromoethyloxy]-isocoumarin

We mixed the ethyl 2-bromo homopthalate prepared as described above (4g) together with phosphorous pentachloride (8.2 g) and benzene (100 ml).The mixture was refluxed for 4.5 hours, filtered hot and evaporated invacuo. The reddish-brown oily residue was chromatographed immediately ona 24 mm×175 mm silica gel column using dichloromethane as eluant.Fractions of 20 ml were collected and assayed by TLC. The4-chloro-3-[2-bromoethyloxy]-isocoumarin eluted in fractions 2-6. Thefractions were pooled, evaporated in vacuo and the resulting residue wasrecovered as a clear, light yellow oil (2.2 g).

4-chloro-3-[3-oxypropanoic acid]-isocoumarin

The 4-chloro-3-[2-bromoethyl]-isocoumarin (1.4 g) prepared above wasdissolved in anhydrous THF and added directly to a refluxing solution ofmagnesium turnings (170 mg), and a few crystals of iodine in 15 mlanhydrous THF, which was stirring under argon. The mixture was refluxedfor 1.5 hours. It was then poured over excess dry ice in a 400 mlbeaker. We let the mixture stand at 20° until all the excess CO₂ hadsublimed and then added approximately 100 ml each of diethyl ether andTHF to the mixture which produced a yellow solution containing a largeamount of white, coarse precipitate.

We bubbled anhydrous HCl through this mixture at 20°, which dissolvedmost of the precipitate The solution was then filtered and evaporated invacuo to obtain the crude product. This was then recrystallizedovernight from DCM.

The resulting 4-chloro-3-[3-oxyproponoic acid]-isocoumarin is coupled toa glycine benzyl ester and the resulting product catalyticallyhydrogenated over palladium-carbon, as described in Example 25. Thispseudodipeptide is then coupled to the resin-bound hexadecapeptide,(Gly)₄ --Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu, bystandard peptide synthesis techniques.

EXAMPLE 29 Synthesis of Hirulog-30

Hirulog-30 has the formula:4-chloro-3-[2-aminoethanol]-isocoumarin-(Gly),--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu.The hexadecapeptide, (Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu issynthesized as previously described and left bound to the resin.

The 4-chloro-3-[2-aminoethanol]-isocoumarin moiety is prepared by aprocedure analogous to that described in Example 28 for synthesizing4-chloro-3-[2-bromoethanol]-isocoumarin, except that 2-aminoethanol isused instead of 2-bromoethanol in the initial step of esterifyinghomopthalic acid.

The urea linkage is formed by reacting the amino group of4-chloro-3-[2-aminoethanol]-isocoumarin with the activating agent,carbonyldiimidazole ("CDI"). The resulting intermediate imidazolide isnot isolated, but is reacted with the resin-linked hexadecapeptide toproduce Hirulog-30. Hirulog-30 is then deprotected, cleaved from theresin and purified by the techniques described in Example 4.

EXAMPLE 30 Synthesis of Hirulog-31

Hirulog-31 has the formula argipidyl-(Gly)₅--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH. Wesynthesized the hexadecapeptide (Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu by thestandard peptide synthesis techniques described previously and leave thepeptide bound to the resin. The argipidylglycine portion of this Hirulogis synthesized by the reaction scheme depicted and described below.##STR11##

A Dehydro-N^(g) -NitroArgipidine is synthesized essentially by themethod for synthesizing argipidine, which is described in U.S. Pat. No.4,258,192, herein incorporated by reference. The only differences arethat the guanidinium group is protected by-a nitro function and theheterocyclic ring of the quinoline remains unsaturated. Thisintermediate is used to acylate t-butyl glycine by the method describedin Example 21. The t-butyl ester is removed by standard acid hydrolysistechniques. The resulting free acid is reacted with the hexadecapeptideusing standard coupling techniques. The resultant peptide isdeprotected, cleaved from the resin and purified by the techniquesdescribed in Example 4.

The peptide is then subjected to the hydrogenation procedure describedin the 4,258,192 patent and purified by the HPLC technique described inExample 4.

EXAMPLE 31 Synthesis of Hirulog-32

Hirulog-32 has the formula: H--(D--Phe)--Pro--Arg--(Gly)₅--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH.Hirulog-32 was synthesized, purified and characterized using the methodsdescribed in Example 4, except that BOC-glycine was used instead ofBOC-proline in the cycle following the two cycles of BOC-glycylglycineaddition.

EXAMPLE 32

Synthesis of Hirulog-33

Hirulog-33 has the formula:N-acetyl--Gly--Asp--Phe--Leu--Ala--Glu-(Gly)₃ -Val--Arg--Pro--(Gly)₄--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH.Hirulog-33 was synthesized, purified and characterized by the standardpeptide synthesis techniques employed in Example 4, with appropriateBOC-amino acid substitutions. The CSDM portion of Hirulog-33 has anamino acid sequence that is identical to a segment of the fibrinopeptideA sequence of the Aα chain in human fibrinogen.

EXAMPLE 33

Cleavage of Various Hirulogs by Thrombin

Inhibition of thrombin by Hirulog-8 was found to be transient due to theslow cleavage of the Arg--Pro bond by thrombin. Following this cleavage,thrombin was observed to recover full hydrolytic activity toward achromogenic substrate. Therefore, Hirulog-8 was characterized as a"slow-substrate" inhibitor of thrombin.

The cleavage of Hirulog-8, as well as other Hirulogs of this invention,by human α-thrombin was demonstrated in in vitro assays. Reactionmixtures containing human α-thrombin (1.6 nM) and varying concentrationsof either Hirulog-8, Hirulog-10, Hirulog-18a, Hirulog-18b, Hirulog-18c,Hirulog-19, Hirulog-32 or Hirulog-33 (80 to 160 nM) were prepared in 20mM Tris-HCl, pH 7.4 containing 0.1M NaCl. Aliquots (0.975 ml) of thereaction mixtures were removed at various times and mixed in a cuvettewith 0.025 ml Spectrozyme TH (11 μM final concentration), a chromogenicsubstrate. The initial rate of reaction was determined and, based oncontrol mixtures containing thrombin in the absence of Hirulog, the %inhibition was calculated.

An alternate method employed reverse-phase HPLC separation of aliquotsfrom a Hirulog/thrombin reaction mixture. In this assay we added humanathrombin (0.25 μM final concentrations) to a reaction vessel containingone of the above Hirulogs (12.5 μM final concentration). Aliquots (50μl) were removed both prior to and at various times following theaddition of thrombin. The aliquots were either flash frozen or injecteddirectly onto the HPLC column. The HPLC system employed an AppliedBiosystems Liquid Chromatography System equipped with an Aquapore C,column (0.46×10 cm). The column was equilibrated in 70% solvent A (0.1%TFA in water) and 30% solvent B (0,085% TFA/70% acetonitrile) anddeveloped with a linear gradient of from 30 to 50% solvent B over 30minutes at a flow rate of 1 ml/minute. The effluent stream was monitoredat 214 nm. Peptide concentrations were determined by measurement of peakheights.

Both of the above-described assays allow determination of the rate ofHirulog hydrolysis by thrombin (expressed in M/min) and turnover rate(k_(cat) ; expressed in min⁻¹). Both methods produced comparable k_(cat)values, which are shown in the table below.

    ______________________________________                                                     P.sub.1 -P.sub.1 '                                               INHIBITOR    SEQUENCE        k.sub.cat (min.sup.-1)                           ______________________________________                                        Hirulog-8    Arg-Pro         0.31-0.5                                         Hirulog-10   Arg-Sar         10                                               Hirulog-18a  β-HomoArg-Gly                                                                            <0.01                                            Hirulog-18b  β-HomoArg-Pro                                                                            <0.01                                            Hirulog-18c  β-HomoArg-Val                                                                            <0.01                                            Hirulog-19   Arg[psiCH.sub.2 NH]-Gly                                                                       <0.01                                            Hirulog-32   Arg-Gly         535                                              Hirulog-33   Arg-Pro         0.056                                            ______________________________________                                    

As shown above, Hirulog-8, -10, -32 and -33 were cleaved by thrombinwith k_(cat), values ranging from 0.056 min⁻¹ (slow cleavage) to 535min⁻¹ (fast cleavage). In contrast, Hirulog-18a, -18b, -18c, and -19appear to be resistant to thrombin cleavage.

FIG. 5, panels A and B, show a more detailed analysis of the cleavage ofHirulog-8 by thrombin. As depicted in FIG. 5, panel A, concentrations ofHirulog-8 in slight excess over thrombin exhibited a transientinhibitory activity (greater than, or equal to, 10 minutes, depending onthe Hirulog concentration). Progressively higher concentrations ofHirulog-8 demonstrated prolonged inhibitory effects. A linearrelationship between duration of inhibition and Hirulog-8 concentrationis shown in FIG. 5, panel B. From these data, we calculated a turnovertime, or k_(cat) of 0.37 min⁻¹.

By purification and sequence analysis of the Hirulog-8-derived digestionproducts produced in the reactions above, we determined that Hirulog-8was slowly cleaved by thrombin at the Arg--Pro bond. This is a highlyunusual cleavage site for serine proteases and we believe it to besusceptible to cleavage in Hirulog-8 due to the high affinity of thepeptide for thrombin.

EXAMPLE 34 The Effect of Linker Length on the Activity of Hirulog

Hirulog-8, Hirulog-13, Hirulog-15, and Hirulog-16 differ from oneanother only by the length of the polyglycine portion of theirrespective linker segments in order to determine what effect linkerlength has on activity, we compared the inhibition of human α-thrombinby each of these Hirulogs. The following table lists the linker lengthsof each of these Hirulogs:

    ______________________________________                                        Peptide      Linker Length (Å)                                            ______________________________________                                        Hirulog-8    24                                                               Hirulog-13   18                                                               Hirulog-15   30                                                               Hirulog-16   36                                                               ______________________________________                                    

The antithrombin activities of these Hirulogs was measured towardthrombin-catalyzed hydrolysis of Spectrozyme TH essentially as describedin Example 9. FIG. 6 depicts the relationship of linker length to K_(i)for Hirulog inhibition of this thrombin-catalyzed reaction. This figureshows that Hirulogs-8, -15 and -16 have comparable inhibitoryactivities, while Hirulog-13, with an 18Å linker length, has an activityreduced by more than 10-fold. This confirms that linker lengths of >18Åand <42Å do not affect Hirulog activity. While not wishing to be boundby theory, applicants believe this is due to the fact that the Hiruloglinker is equally disordered when free in solution as when bound tothrombin. Applicants also believe that there is little cooperativity inthe binding of the CSDM and ABEAM portions of the thrombin inhibitors ofthis invention to thrombin.

EXAMPLE 35 Inhibition of Thrombin-Catalyzed Hydrolysis by VariousHirulogs

We compared the inhibitory activity of various thrombin inhibitors ofthe present invention on thrombin-catalyzed hydrolysis of atripeptidyl-p-nitroanilide substrate. The antithrombin activities ofHirulog-10, Hirulog-18a, Hirulog-18b, Hirulog-18c, Hirulog-19,Hirulog-32 and Hirulog-33 were assayed by the method described inExample 9, using Spectrozyme TH as a substrate. The table below liststhe calculated K_(i) values as well as the P₁ --P₁ ' sequence, of eachof these thrombin inhibitors.

    ______________________________________                                                     P.sub.1 -P.sub.1 '                                               INHIBITOR    SEQUENCE         K.sub.i (nM)                                    ______________________________________                                        Hirulog-8    Arg-Pro          1.9 ± 1.4                                    Hirulog-10   Arg-Sar          >2,000                                          Hirulog-18a  β-HomoArg-Gly                                                                             7.4                                             Hirulog-18b  β-HomoArg-Pro                                                                             4.6                                             Hirulog-18c  β-HomoArg-Val                                                                             205.0                                           Hirulog-19   Arg[psiCH.sub.2 NH]-Gly                                                                        20.0                                            Hirulog-32   Arg-Gly          >2,000                                          Hirulog-33   Arg-Pro          3.6                                             ______________________________________                                    

As indicated above, Hirulog-10 and Hirulog32 were poor inhibitors ofthrombin-catalyzed hydrolysis of Spectrozyme TH. This was consistentwith the fact that each of these inhibitors was rapidly cleaved bythrombin at the P₁ --P₁ ' bond in Hirulog-19, wherein this bond wasreduced to the psiCH₂ --NH linkage and rendered non-cleavable bythrombin, effective inhibition of thrombin hydrolysis was observed.

The studies with β-homoarginine-containing inhibitors (Hirulogs-18a,-18b and -18c) demonstrated that this amino acid derivative may replacearginine in the inhibitors of this invention without affecting activity.Moreover, this shows that displacement of the amide bond by onemethylene does not markedly reduce thrombin inhibitory activity. The 30-to 50fold increase in K_(i) for Hirulog-18c, as compared to Hirulog-18aand -18b, respectively, suggests that the structure of the P'₁ aminoacid is important in inhibitory activity. Without wishing to be bound bytheory, applicants believe that the presence of phi-psi angles in theP'₁ amino acid (Gly in Hirulog-18a; Pro in Hirulog-18b) as well asconformational constraints, (such as is caused by the proline inHirulog-18b) contribute to the potency of the inhibitors of thisinvention. An alternate possibility is that the β-branched side chain ofthe P'₁ amino acid Val in Hirulog-18c may impair binding of the CSDMportion of that molecule to the thrombin reactive center due to stericconsiderations.

EXAMPLE 36 Binding of Hirulog-8 to the Active Site of Thrombin

Diisopropylfluorophosphate (DFP) is a well-known inhibitor of serineproteases, including thrombin, which acts by covalently modifying thehydroxyl group of Ser-195. We added a 270-fold excess of ¹⁴ C-DFP tothrombin, in 0.1M sodium borate, pH 8.0. Following a 10 minute reaction,formation of a thrombin complex was demonstrated by SDS-PAGE andfluorographic analyses (FIG. 7, lane 1). When the reaction was performedin the presence of Sulfo--Tyr₆₃ -N-acetylhirudin₅₃₋₆₄ (at 300 and3000-fold molar excess over thrombin), the modification of thrombin by[¹⁴ C]-DFP was not altered significantly (FIG. 7, lanes 4 and 5).However, when we performed the reaction in the presence of Hirulog-8 (at3- or 30-fold molar excess over thrombin) the incorporation of [¹⁴C]-DFP into the thrombin catalytic site was completely blocked (FIG. 7,lanes 2 and 3). These data demonstrate that the CSDM of the thrombininhibitors of this invention are capable of binding to the catalyticsite of thrombin and inhibiting catalytic activity.

EXAMPLE 37 Comparison of Antithrombin Activity of Hirulog-8 and aSynthetic Catalytic Site Directed Pentapeptide(D--Phe--Pro-Arg--Pro--Gly)

As shown in FIG. 1, Hirulog-8, unlike its constituent anion-bindingexosite associating moiety, Sulfo-Tyr₆₃ -N-acetyl-hirudin₅₃₋₆₄ was ableto inhibit thrombin-catalyzed hydrolysis of small p-nitroanilidesubstrates. Similarly, we have tested the ability of a(D--Phe)--Pro--Arg--Pro--Gly pentapeptide to inhibit thrombin catalyticreactivity.

The pentapeptide was synthesized as described in Example 4, using aBOC-glycine-divinylbenzene resin. The pentapeptide was purified andcharacterized as described in Example 4.

The effects of both Hirulog-8 and this pentapeptide towardthrombin-catalyzed hydrolysis of Spectrozyme TH were studied asdescribed in Example 9, using fixed peptide concentrations of 50 nM or10 μM, respectively. Our results show that while nanomolarconcentrations of Hirulog-8 can inhibit the thrombincatalyzed reaction,concentrations of pentapeptide as high as 10 μM have no significanteffect on the thrombin-catalyzed rate. These data show that the CSDMcomponent of the thrombin inhibitors of this invention is, by itself,only a weak inhibitor of thrombin's catalytic function.

EXAMPLE 38 In Vivo Anticoagulant Activity of Hirulog-8

We determined the in vivo anticoagulant activity of Hirulog-8 followingintravenous administration of this peptide into baboons. We used variousdosages of Hirulog-8 ranging from 0.002 to 0.2 mg/kg/min. Baboons (male,10-15 kg) were sedated with ketamine hydrochloride prior toadministration of Hirulog-8. Whole blood from treated and controlanimals was removed from a catheter placed in the femoral vein andcollected into 3.8% sodium citrate (9:1; blood:sodium citrate). Plasmawas obtained by standard methods and the APTT was recorded by methodsdescribed in Example 10. As shown in FIG. 8, Hirulog-8 yielded adose-dependent increase in the APTT. A 200% increase in the APTT(considered a therapeutic range) was achieved with the lowest Hirulogdose (0.002 mg/kg/min. infusion).

EXAMPLE 39 Inhibition of Clot-Bound Thrombin by Hirulog-8

It is known that thrombin can bind to a fibrin clot and, once absorbed,continue to cleave additional fibrinogen, resulting in growth of theclot. Clot-bound thrombin has been shown to be resistant toneutralization by the heparin-anti-thrombin III complex [P. J. Hogg etal., "Fibrin Monomer Protects Thrombin From Inactivation ByHeparin-Antithrombin III: Implications for Heparin Efficacy", Proc.Natl. Acad. Sci. USA, 86, pp. 3619-23 (1989)], but may be inhibited byantithrombin III-independent inhibitors, such as PPACK, hirudin orSulfo-Tyr₆₃ -N-acetyl-hirudin₅₃₋₆₄. Clot-bound thrombin is believed toplay a role in thrombus accretion and in rethrombosis followingthrombolytic therapy.

We compared the abilities of Hirulog-8 and heparin to inhibit clot-boundthrombin using the method described by J. I. Weitz et al., "Clot-BoundThrombin Is Protected from Heparin Inhibition--A Potential Mechanism forRethrombosis After Lytic Therapy", Blood, 74, p. 136a, (1989).

A clinically relevant dose of heparin (0.1 U/ml) inhibitedfibrinopeptide A (FPA) release catalyzed by soluble thrombin byapproximately 70%. However, a similar dose had no effect on FPA releasecatalyzed by clot-bound thrombin. In contrast, Hirulog-8 had an almostidentical inhibitory effect on FPA release catalyzed by either solubleor clot-bound thrombin (FIG. 9).

This study indicated that Hirulog-8, as well as the other thrombininhibitors of this invention, are more effective than current drugs inblocking thrombus accretion, increasing the rate of thrombolyticreperfusion and preventing rethrombosis following thrombolytictreatment.

EXAMPLE 40 The Effect of Hirulog-8 on In Vivo Platelet-DependentThrombosis

Because baboons are known to have similar coagulation and hemostaticresponses as man, we utilized a baboon model to determine the ability ofHirulog-8 to interrupt platelet-dependent thrombosis. Specifically, weplaced various thrombogenic surfaces in a chronic exteriorized AV shuntin the animals. These surfaces included segments of endarterectomizedbaboon aorta, collagen-coated silastic tubing, collagen-coated Gortexand Dacron vascular graft. Following placement in the shunt, thesurfaces were exposed to native flowing blood to elicit thrombusformation. We measured the formation of thrombi over a period of 60minutes by monitoring the deposition of platelets on the thrombogenicsurface. These measurements were recorded by external gamma-cameraimaging following pre-infusion of the test animal with autologous ¹¹¹In-labeled platelets.

Placement of a 5 cm segment of endarterectomized baboon aorta in theexteriorized AV shunt in the absence of Hirulog-8 led to atime-dependent deposition of platelets. This accumulation reached aplateau in 60 minutes, at which time a total of 14.0±5.0×10⁸platelets/cm were found deposited on the endarterectomized segment.Doses of 0.002 and 0.01 mg/kg/min of Hirulog-8 inhibited plateletdeposition by 53.6% and 75.5%, respectively. These results are depictedin FIG. 10. The ED₅₀ for Hirulog-8 (the dosage required to reduceplatelet deposition by 50%) in this model system was 0.002 mg/kg/min.

When we placed 5 cm segments of collagen-coated silastic tubing in theAV shunt, 12.6±5.0×10⁸ platelets/cm were deposited after 60 minutes inthe absence of Hirulog-8. Administration of Hirulog-8 resulted in adose-dependent inhibition of platelet deposition. A dosage of 0.04mg/kg/min Hirulog-8 completely inhibited platelet deposition. Theresults of this portion of the experiment are depicted in FIG. 11. TheED₅₀ of Hirulog-8 in this system was calculated to be 0.004 mg/kg/min.

Both collagen-coated Gortex or Dacron vascular grafts are known to bemore thrombogenic than silastic tubing. A total of 35.0±6.0×10⁸platelets/cm were deposited on the Gortex following a 60 minute exposureto native blood in the absence of Hirulog-8. We found that Hirulog-8once again demonstrated a dose-dependent antithrombotic effect towardsplatelet thrombus formation. A dose of 0.2 mg/kg/min Hirulog-8 caused a62.9% inhibition of platelet deposition. The ED₅₀ for Hirulog-8 in theGortex system was 0.135 mg/kg/min. A similar result was obtained forDacron grafts. The higher dosage of Hirulog-8 required to inhibitplatelet deposition on these two surfaces was to be expected because oftheir high thrombogenic activity.

We also determined the effect of Hirulog-8 toward both high and lowshear platelet-dependent thrombus formation using a dual-chamber device,which allowed for simultaneous measurements of both shear conditions.The device was comprised of a 2 cm segment of collagen-coated Gortexfollowed by 2 cm segments of expanded diameter. Using this device,thrombus formation was initiated by exposure of native flowing blood toa segment of the collagen-coated Gortex at high shear. This part of theexperimental protocol simulated arterial-like conditions. When the bloodentered the expanded diameter segments, low-shear, vortex conditionswere maintained, thereby simulating venous thrombosis in controlanimals, a total of 9.3 ±2.3×10⁸ and 6.1±0.5×10⁸ platelets/cmaccumulated after 40 minutes in the high and low shear segments,respectively. Hirulog-8 inhibited platelet deposition in both high andlow shear segments in a dose-dependent fashion. A dose of 0.05 mg/kg/mininhibited platelet accumulation by 42.6% at low shear and by 29.0% athigh shear.

EXAMPLE 41

Comparison of Hirulog-8 With Other Anti-Thrombotic Agents in InhibitingAcute Platelet-Dependent Thrombosis

We examined the effects of heparin, low molecular-weight heparin andrecombinant hirudin on platelet deposition in the collagen-coatedsilastic tubing/exteriorized AV shunt baboon model described in Example40.

It has previously been shown that heparin administered as a 160 U/kgbolus injection followed by a 160 U/kg/hr infusion inhibited plateletdeposition to a level of about 80% of that observed in a saline-treatedcontrol animal. Low molecular-weight heparin, given as a bolus injectionof 53 anti-Xa U/kg, followed by infusion at 53 anti-Xa U/kg/hr, yieldedsimilar results [Y. Cadroy, "in Vivo Mechanism of Thrombus FormationStudies Using a Primate Model", Doctoral Thesis, L'Universite PaulSabatier de Toulouse (Sciences) (1989)]. At equivalent molar doses (5nmole/kg/min), recombinant hirudin [A. B. Kelly et al., "RecombinantHirudin Interruption of Platelet-Dependent Thrombus Formation",Circulation, 78, p. II-311 (1988)]and Hirulog-8 both inhibitedplatelet-dependent thrombus formation by 60-70% as compared to thecontrol. These results are depicted in FIG. 12. Other thrombininhibitors have previously been tested in the baboon model [A. B. Kelleyet al., "Comparison of Antithrombotic and Antihemostatic EffectsProduced by Antithrombins in Primate Models of Arterial Thrombosis",Thromb. and Hemostas., 62, p. 42 (1989)]. The reported ED₅₀ doses oncollagen-coated surfaces for those agents, as well as our ED₅₀determinations, are summarized in the table below:

    ______________________________________                                        Agent          ED.sub.50                                                      ______________________________________                                        PPACK          75         nmoles/kg/min                                       Gyki 14,451    500                                                            Benzamidine    3000                                                           Argipidine (MD805)                                                                           550                                                            rec-Hirudin    <5                                                             Hirulog-8      <5                                                             ______________________________________                                    

EXAMPLE 42 The Effect of Hirulog-8 on Fibrin Deposition

We measured the effect of Hirulog-8 on the deposition of fibrin(ogen) inthe thrombi formed in the endarterectomized aortic and collagen-coatedsilastic tubing segments model systems described in Example 40. Fibrindeposition was determined by measurement of ¹²⁵ I-fibrin(ogen) 30 daysafter completion of the ¹¹¹ In-platelet assay described above. Thisallowed the label to decay to a non-interfering level.

FIG. 13 demonstrates that in the absence of Hirulog-8, 0.17 mg/cm fibrinwas deposited on the collagen-coated tubing following the 60 minuteexposure to flowing blood described in Example 40. Doses of 0.01 and0.04 mg/kg/min completely inhibited fibrin(ogen) deposition. Similarresults were obtained with the endarterectomized aortic segment model.These results show that the thrombin inhibitors of this invention areeffective in reducing fibrin(ogen) deposition associated with athrombus, as well as blocking acute platelet-dependent thrombusformation.

EXAMPLE 43

Measurement of Clearance Times For Hirulog-8

We used a baboon model to determine Hirulog-8 clearance times followingintravenous infusion, single bolus intravenous injection and singlebolus subcutaneous injection. APTT assays, performed as described inExample 11, were used to monitor clearance times.

We administered various dosages of Hirulog-8 (0.002-0.2 mg/kg/min) tobaboons via systemic intravenous infusion, over a period of 60 minutes.APTT was measured following the 60 minute infusion and at various timeintervals thereafter. We determined the average half-time for Hirulog-8clearance to be 9.2 ±3.3 minutes.

To determine clearance time after a single bolus injection, we injectedbaboons with a dose of 1 mg/kg Hirulog-8 intravenously orsubcutaneously. APTT measurements were taken at various time intervalsfollowing injection. FIG. 14 demonstrates that APTT increased to a peakof 570% of control value 2 minutes after intravenous injection. Thehalf-life of Hirulog-8 following intravenous injection was 14 minutes.

FIG. 15 demonstrates that at the earliest time point followingsubcutaneous injection of Hirulog-8 (i.e. 15 minutes), APTT wasincreased to approximately 200% of control. Clearance via thesubcutaneous route was prolonged to a half-time of 340 minutes.Hirulog-8 administered subcutaneously was found to be quantitativelyadsorbed.

EXAMPLE 44

Effect of Hirulog-8 in Baboon Models of Disseminated IntravascularCoagulation

We induced septicemia in baboons by injection of a lethal dose of liveE. coli according to the method described by F. B. Taylor et al. J.Clin. Invest., 79, pp. 918-25 (1987). Hirulog-8 was infused at a dose of0.08 mg/kg/hr from 15 minutes prior to the injection of E. coli to up to6 hours following injection. In the absence of Hirulog-8, E. coliinduced septic shock led to a marked decline in neutrophil count, bloodpressure and hematocrit. Control animals displayed a reduction inhematocrit to 70% of baseline and a drop in blood pressure to 20% ofbaseline after 3 hours. Administration of Hirulog-8 completelyattenuated hematocrit drop and limited the peak drop in blood pressureto 60% of baseline.

Despite attenuation of DIC by Hirulog-8, the lethal infusion of E. colistill resulted in morbidity. An autopsy of both control andHirulog-8-treated animals revealed massive tissue edema in both groups.However, only the control group displayed intravascular thrombosis. Theresults of the autopsies show that interruption of the coagulopathicstage of septicemia alone is not sufficient to prevent morbidity due toseptic shock.

EXAMPLE 45 Effect of A Combination of tPA and Hirulog-8 on Thrombolysis

To determine the effect of Hirulog-8 on potentiating tPA-inducedthrombolysis, we used a rat model for arterial thrombolysis. In thismodel, an experimental thrombus was formed in the abdominal aortafollowing balloon catheter denudation and high grade (95%) stenosis.Blood flow and blood pressure were recorded distal to the site of injuryand stenosis. We randomized the rats to received tPA (1.0 mg/kg bolusfollowed by 1.0 mg/kg/hr infusion) together with one of the following:saline, heparin (10 U/kg bolus, followed by 1.5 U/kg/min infusion),recombinant hirudin (1.0 mg/kg bolus followed by 0.02 mg/kg/hr infusion)or Hirulog-8 (0.6 mg/kg bolus followed by 0.02 mg/kg/hr infusion). Theantithrombotic agent or saline was administered concomitant with tPA andfor an additional 50 minutes following the end of tPA infusion.

FIG. 16 depicts the results of these experiments. Animals treated withtPA+saline exhibited reperfusion times of 16.2 minutes. Heparin reducedreperfusion time to 12.2 minutes, while recombinant hirudin reduced itto 13.0 minutes. Neither of these decreases were statisticallysignificant (p<0.05). The combination of Hirulog-8 with tPAsignificantly reduced reperfusion time to 4.4 minutes (p<0.01), thusaccelerating the fibrinolytic effect of tPA by a factor of four.

Heparin, hirudin and Hirulog-8 all significantly prevented reocclusionas compared to saline-treated controls (FIG. 17). Each of these agentsalso prolonged APTT to values of 600%, 500% and 400%, respectively, overcontrol values (FIG. 18). Finally, each of heparin, hirudin andHirulog-8 increased the time of vessel patency to values of 80.2%, 82%and 93.1%, respectively (control=43.6%) (FIG. 19). These resultsdemonstrate that the thrombin inhibitors of the present invention aresuperior to other known anti-thrombotics in increasing the efficacy oftPA.

EXAMPLE 46 Effect of Hirulog-8 and Other Antithrombotic Agents onBleeding Times in Baboons

We employed the template bleeding time measurement to examine theeffects of Hirulog-8 on hemostasis.

Various dosages of Hirulog-8 (0.002 to 0.2 mg/kg/min) were analyzed fortheir effect on bleeding time. Doses of 0.002 to 0.04 mg/kg/min causedno significant increase in bleeding times. The results of thisexperiment are depicted in FIG. 20. At a dose of 0.1 mg/kg/min,Hirulog-8 causes a two-fold increase in bleeding time over controlvalues. At 0.2 mg/kg/min Hirulog-8, bleeding times increased to 3 timescontrol values. These results clearly demonstrate that dosages requiredto inhibit platelet-dependent thrombosis (0.002 mg/kg/min; see Example40) do not cause a significant effect on hematostatic plug formation.

We also tested the effects of a variety of other agents on templatebleeding time in the baboon, as well as on systemic anticoagulanteffects (as measured by APTT). These results are summarized below:

    ______________________________________                                                         APTT      Bleeding                                           Agent            (% control)                                                                             Time (min)                                         ______________________________________                                        Hirulog-8        300.6     5.5                                                rec-hirudin      393.9     12.1                                               PPACK            287.9     12                                                 Gyki 14,451      439.4     14                                                 Benzamidine      757.6     10                                                 Argipidine (MD805)                                                                             >900      >30                                                Heparin          706.1     10                                                 ______________________________________                                    

EXAMPLE 47 Synthesis of Hirulog-34

Hirulog-34 has the formula:H--(D--Phe)--Pro--Arg--(tetraethyleneglycolylsuccinyl)--Asn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu--OH.The decapeptideAsn--Gly--Asp--Phe--Glu--Glu--Ile--Pro--Glu--Glu--Tyr--Leu issynthesized as previously described and left bound to the resin. Theremaining portion of the molecule is synthesized by the reaction schemedepicted and described below: ##STR12##

N.sup.α -BOC-(D--Phe)--Pro--N^(g), N^(g) --(Cbz)₂ --Arg

N.sup.α -BOC-N^(g) -(Cbz)₂ --Arg (Bachere, Inc., Torrance, Calif.) isreacted with excess ethereal diazomethane and then treated with acid toremove the N.sup.α -BOC group. The resulting product, N^(g),N^(g)-(Cbz)₂ -Arginine methyl ester is then dissolved in DMF, cooled in anice bath and treated with, in order, 2 equivalents of N.sup.α-BOC-proline, 1 equivalent of butanol, 1 equivalent of EDCI and 1equivalent of diisopropylethylamine. The reaction mixture is stirredovernight, then diluted with 5 volume of cold water and extracted withethyl acetate. The organic phase is recovered and washed successivelywith equal volumes of saturated citric acid, saturated NaHCO₃ andsaturated NaCl. The product is then dried over MgSO₄ and concentrated invacuo. The resulting dipeptide intermediate, N.sup.α-BOC-prolyl-N^(g),N^(g) -(Cbz)₂ -Arginine methyl ester, is treated witha 10-fold molar excess of 4N HCl/dioxane for 30 minutes. The free HCl isremoved in vacuo and the residue dissolved in anhydrous DMF. The productis then cooled in an ice bath and reacted with 2 equivalents of N.sup.α-BOC-(D--Phe) in the presence of butanol, EDCI anddiisopropylethylamine, as described above. The crude, orthogonallyprotected tripeptide is isolated as described above and purified on asilica gel column eluted with chloroform:methanol (95:5) containing 0.1%NH₄ OH. The N.sup.α -BOC-(D)-phenylalanylprolyl-N^(g),N^(g) -(Cbz)₂-Arginine methyl ester is then saponified with 2 equivalents of LiOH inmethanol:water (2:1) at room temperature for 3 hours. The methanol isremoved in vacuo and the aqueous solution washed with 2 volumes ofdiethyl ether. The solution was then acidified to pH 3 with saturatedcitric acid. The resulting crude tripeptide free acid is extracted intoethyl acetate. The organic phase is washed with 3 volumes of saturatedNaCl, dried over anhydrous MgSO₄ and concentrated in vacuo. Theresulting N.sup.α -BOC-(D)-phenylalanylprolyl-N^(g),N^(g) -(Cbz)₂--Arginine is purified by reverse-phase HPLC under conditions describedin Example 4.

N.sup.α -BOC-(D)--Phenylalanylprolyl-N^(g),N^(g) -Cbz)₂ -ArginineTetraethyleneglycol Ester

A solution of the above tripeptide is dissolved in THF and esterifiedwith 1.5 equivalents of tetraethylene glycol in the presence of 1equivalent each of diethylazodicarboxylate and triphenylphosphine asdescribed in O. Mitsunobu, "The Use of DiethylAzodicarboxylate andTriphenylphosphine in Synthesis and Transformation of Natural Products,Synthesis, pp. x1-28 (1981), the disclosure of which is hereinincorporated by reference.

N.sup.α -BOC-(D)Phenylalanylprolyl-N^(g),N^(g) -(Cbz)₂ -ArginineTetraethyleneglycolyl Hemisuccinate

The resulting compound is dissolved in DMF and esterified with 1equivalent of succinic anhydride in the presence of i equivalent ofdiisopropylethylamine. The volatile solvents are removed in vacuo andthe free acid is purified by reverse-phase HPLC under conditionsdescribed in Example 4.

N.sup.α -BOC-(D)phenylalanylprolyl-N^(g),N^(g) -(Cbz)₂ -ArginineTetraethyleneglycolyl Hemisuccinate N-Hydorxysuccinimide Ester

A solution of the above acid is mixed with 1 equivalent ofN-hydroxysuccinimide in DMF, cooled in an ice bath and mixed with 1equivalent of DCC in DMF, added dropwise. The reaction is stirred atroom temperature for 24 hours, filtered to remove the precipitateddicyclohexyl urea and concentrated in vacuo. The solution is thenconcentrated with cold benzene/hexane to give the crude peptide-glycolconjugated N-hydroxysuccinimide ester.

The above compound is reacted with the resin-bound dodecapeptide, asdescribed in. Example 21. The resulting Hirulog-34 is cleaved from theresin, purified and characterized as described in Example 4.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other embodiments which utilize the molecules, compositions,combinations and methods of this invention. Therefore, it will beappreciated that the scope of this invention is to be defined by theclaims appended hereto rather than the specific embodiments which havebeen presented hereinbefore by way of example.

We claim:
 1. A thrombin inhibitor consisting of:a) a catalyticsite-directed moiety that binds to and inhibits the active site ofthrombin wherein said catalytic site-directed moiety is selected fromgeneral serine proteinase inhibitors, heterocyclic protease inhibitors,thrombin-specific inhibitors, transition state analogues, benzamidine,DAPA, NAPAP, argipidine, or moieties of the formulae: X--A₁ --A₂ --A₃--Y or X--C₁ --C₂ --A₃ --Y,wherein X is hydrogen or is characterized bya backbone chain consisting of from 1 to 35 atoms; A₁ is Arg, Lys orOrn; A₂ is a non-amide bond; A₃ is characterized by a backbone chainconsisting of from 1 to 9 atoms; Y is a bond; C₁ is a derivative of Arg,Lys or Orn comprising a carboxylate moiety that is reduced, or displacedfrom the α-carbon by a structure characterized by a backbone chain offrom 1 to 10 atoms; and C₂ is a non-cleavable bond; b) a linker moietycharacterized by a backbone chain having a calculated length of between18Å and 42Å; and c) an anion binding exosite associating moiety;saidcatalytic site-directed moiety being bound to said linked moiety andsaid linker moiety being bound to said anion binding exosite moiety,wherein said inhibitor is labeled with a radioisotope and is capable ofsimultaneously binding to the catalytic site and the anion bindingexosite of thrombin.
 2. The thrombin inhibitor according to claim 1,wherein said anion binding exosite moiety consists of the formula:

    W--B.sub.1 --B.sub.2 --B.sub.3 --B.sub.4 --B.sub.5 --B.sub.6 --B.sub.7 --B.sub.8 --Z;

wherein W is a bond; B₁ is an anionic amino acid; B₂ is any amino acid;B₃ is Ile, Val, Leu, Nle or Phe; B₄ is Pro, Hyp, 3,4-dehydroPro,thiazolidine-4-carboxylate, Sar, any N-methyl amino acid or D--Ala; B₅is an anionic amino acid; B₆ is an anionic amino acid; B₇ is alipophilic amino acid selected from the group consisting Tyr, Trp, Phe,Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of one of theselipophilic amino acids and any amino acid; B₈ is a bond or a peptidecontaining from one to five residues of any amino acid; and Z is acarboxy terminal residue selected from OH, C₁ -C₆ alkoxy, amino, mono-or di-(C₁ -C₄) alkyl substituted amino or benzylamino.
 3. The thrombininhibitor according to claim 2, wherein B₁ is Glu; B₂ is Glu; B₃ is Ile;B₄ is Pro; B₅ is Glu; B₆ is Glu; B₇ is Tyr--Leu, Tyr(SO₃ H)--Leu,Tyr(OSO₃ H)--Leu or (3-, 5-diiodoTyr)--Leu; B₈ is a bond; and Z is OH.4. The thrombin inhibitor according to claim 1, wherein said backbonechain of said linker moiety consists of any combination of atomsselected from the group consisting of carbon, nitrogen, sulfur andoxygen.
 5. The thrombin inhibitor according to claim 4, wherein saidlinker comprises the amino acid sequence:Gly--Gly--Gly--Asn--Gly--Asp--Phe.
 6. The thrombin inhibitor accordingto claim 1, wherein said catalytic site-directed moiety binds reversiblyto and is cleaved by thrombin.
 7. The thrombin inhibitor according toclaim 1, wherein said catalytic site-directed moiety binds reversibly toand cannot be cleaved by thrombin.
 8. The thrombin inhibitor accordingto claim 1, wherein said catalytic site-directed moiety bindsirreversibly to thrombin.
 9. The thrombin inhibitor according to claim1, wherein X is D--Phe--Pro; A₁ is Arg; and A₃ is D--Pro, Pro, or Sar.10. The thrombin inhibitor according to claim 9, wherein said thrombininhibitor is selected from the group consisting of Hirulog-8 andHirulog-12.
 11. The thrombin inhibitor according to claim 1, wherein Xis N-acetyl--Gly--Asp--Phe--Leu--Ala--Glu--Gly--Gly--Gly--Val; A₁ isArg; and A₃ is Pro.
 12. The thrombin inhibitor according to claim 1,selected from the group consisting of Hirulog-18a and Hirulog-18b.
 13. Acomposition for imaging of a fibrin or a platelet thrombus in a patient,said composition comprising a pharmaceutically acceptable buffer and athrombin inhibitor according to any one of claims 1 or 2-12.
 14. Amethod for imaging a fibrin or a platelet thrombus in a patientcomprising the steps of:a) administering to said patient a compositionaccording to claim 13; and b) using detecting means to observe thethrombin inhibitor present in said composition.
 15. The thrombininhibitor according to any one of claims 1 to 12, wherein saidradioisotope is selected from the group consisting of ¹²³ I, ¹²⁵ I and¹¹¹ n.
 16. A composition for imaging of a fibrin or a platelet thrombusin a patient, said composition comprising a pharmaceutically acceptablebuffer and a thrombin inhibitor according to claim
 15. 17. The methodaccording to claim 14 wherein said patient is a human.